Lipid oxidation pathways and nitric oxide signaling pathways are interrelating pathways with significance both separately and in conjunction (references 1-2). Lipid oxygenating pathways involve the formation of hydroperoxides, alkanes, alkenes, aldehydes (notably nonenal), epoxides, alcohols and other species from polyunsaturated fatty acids that are readily oxidized. The nitric oxide signaling pathway has been of great interest in the last several years and involves the formation of nitric... Show moreLipid oxidation pathways and nitric oxide signaling pathways are interrelating pathways with significance both separately and in conjunction (references 1-2). Lipid oxygenating pathways involve the formation of hydroperoxides, alkanes, alkenes, aldehydes (notably nonenal), epoxides, alcohols and other species from polyunsaturated fatty acids that are readily oxidized. The nitric oxide signaling pathway has been of great interest in the last several years and involves the formation of nitric oxide near the site of inflammation and its transport to other tissues to function as a messenger. Oxidized lipid signaling pathways are an active area of research, and many interactions with the nitric oxide pathway are left open to discussion. The interactions of nitric oxide and lipid oxidizing enzymes have been demonstrated, and these interactions are of particular significance in the regulation of vascular homeostasis. The studies presented here investigate the specific interactions of soybean lipoxygenase-1, a lipid-oxygenating enzyme, and nitric oxide by electron paramagnetic resonance (EPR) analyses of single lipoxygenase crystals complexed with nitric oxide. Nitric oxide is known to bind with high affinity to the lipoxygenase active site iron. Though nitric oxide is often used as a dioxygen analog, molecular oxygen binds to a fatty acid radical and not directly to the active site iron in the lipoxygenase mechanism, so nitric oxide was not used as a dioxygen analog in our studies. Rather, we expected the electron configuration and any differences in coordination to be a reasonable model of a transition state that mimics a peroxyl radical formed during catalysis. The aim of this project was to determine the local structure of the active site of a transition state analog with mechanistic significance. To complete this line of experimentation, I obtained a number of lipoxygenase crystals similar to those used for x-ray analysis. The crystals were then complexed with nitric oxide using protocols that are similar to those used in previous studies of lipoxygenase and other iron proteins. X-band (9.26 GHz) EPR experiments were performed and analyzed to determine the suitability of the experimental methodology presented here for observation of changes in the electronic orbital structure of the active site. Differences between the structure of the resting enzyme and the structure of the NO–LOX complex that may be observed with EPR include both the positions of the electron orbitals and the spatial orientation of the nitric oxide-iron bond, from which one may be able to infer the positions and coordination of the other iron ligands. While no conclusions about the electronic structure of the lipoxygenase iron-nitric oxide bond could be drawn from these experiments, the suitability of the experimental conditions for further studies was proven. This project also represents an advancement in the area of EPR studies of small protein crystals, similar in size to those used in x-ray diffraction experiments. Further studies of the complex that were not included in the masters project may include W-band (92.4 GHz) EPR studies and X-ray crystallography of crystals of the nitric oxide-lipoxygenase complex. Show less

Date Issued

2004

Identifier

FSU_migr_etd-2421

Format

Thesis

Title

ABA Contents in the Guard-Cell Symplast and Guard-Cell Apoplast Are Not Correlated with Stomatal Aperture Size under Three Conditions of Water Sufficiency.

Guard cells respond to environmental stimuli by opening and closing stomata, which balance CO2 uptake and water conservation. Stomatal closure under water deficiency and the involvement of abscisic acid (ABA) in this response are well-known. However, whether ABA plays a role in stomatal regulation under some water-sufficient conditions, such as diurnal changes, humidity shift (i.e. transpiration-rate change) and brief flooding, is not clear. Using an enzyme-linked immunosorbent assay (ELISA)... Show moreGuard cells respond to environmental stimuli by opening and closing stomata, which balance CO2 uptake and water conservation. Stomatal closure under water deficiency and the involvement of abscisic acid (ABA) in this response are well-known. However, whether ABA plays a role in stomatal regulation under some water-sufficient conditions, such as diurnal changes, humidity shift (i.e. transpiration-rate change) and brief flooding, is not clear. Using an enzyme-linked immunosorbent assay (ELISA) with sub-femtomole sensitivity for ABA assays, we studied the relationships of stomatal aperture size with the ABA contents in the symplast and apoplast of guard cells, as well as those in the leaf and the leaf apoplast in Vicia faba under the following three conditions. (1) Diurnal changes. Stomata opened in the morning, reached a maximum opening at 1400 h, and closed at 1800 h. Neither the leaf nor the leaf apoplastic ABA content strongly correlated with stomatal aperture sizes. The ABA contents of the guard-cell compartments did not change over the course of the day, providing evidence that ABA is not involved in diurnal stomatal regulation. (2) Humidity-induced transpiration-rate changes. The transpiration rate of intact plants and that of detached leaves infused with 1µM ABA was decreased by shifting RH from 60% to 90%. The ABA contents of the four compartments were not changed by this humidity shift, in spite of an increase of 2–3 µm in stomatal aperture sizes. Thus, the guard-cell-apoplastic ABA content is not affected by transpiration rate, and ABA may not participate in the stomatal response to transpiration rate. (3) Brief flooding. Stomata closed after brief (4-h) flooding, when the leaf and the leaf apoplastic ABA increased 2–3 fold and the xylem pH increased 0.2 pH units. The leaf ABA increase did not correlate strongly with stomatal aperture size and the xylem ABA delivery rate remained unchanged. The ABA contents in the guard-cell compartments of flooded plants were not different from those of non-flooded plants. Therefore, ABA may not be an initiator of stomatal closure under brief flooding, and xylem alkalinization probably does not induce leaf ABA redistribution to guard cells. Show less

Date Issued

2007

Identifier

FSU_migr_etd-2485

Format

Thesis

Title

Adaptation, Diversification, and Desert Ecology of the Most Diverse Order of Mammals (Mammalia, Rodentia).

Globally, species diversity is regulated by speciation and extinction, and regionally it is regulated by competition, niche, colonization, emigration, and extinction, and more locally, by environmental tolerance and species interactions which filter out non-adapted species based on intrinsic characteristics, or their Hutchinsonian niche. In this dissertation, I examined some of the mechanisms that govern biodiversity patterns in order to determine the main causes of uneven diversity in muroid... Show moreGlobally, species diversity is regulated by speciation and extinction, and regionally it is regulated by competition, niche, colonization, emigration, and extinction, and more locally, by environmental tolerance and species interactions which filter out non-adapted species based on intrinsic characteristics, or their Hutchinsonian niche. In this dissertation, I examined some of the mechanisms that govern biodiversity patterns in order to determine the main causes of uneven diversity in muroid rodent clades, the most diverse superfamily of mammals, comprising 28% of all mammal species. This extensive diversity, in addition to the remarkable eco-morphological adaptability which facilitated their colonization of all terrestrial biomes make muroids an ideal system to study this fundamental question in evolutionary ecology. In addition, the use of robust phylogenies that have recently been developed in muroids and non-muroid rodents makes the order an especially attractive model system to understand the process of mammalian adaptation to arid environments and the ecological interactions that shaped patterns of coexistence within desert communities, the second main goal of the dissertation. The use of a combination of molecular phylogenetics and geometric morphometrics allows for a robust investigation of general patterns that shape the ecological evolution of this group within and without desert habitats, warranting a reinterpretation of classical studies in evolutionary biology, desert ecology, and the traditional systematics of desert rodent clades. Show less

Title: Allergenic cross-reactivity between cashew and pistachio nuts Rationale: Cashew and pistachio belong to Anacardiaceae family and strong allergenic cross-reactivity between nuts of these two species has been reported. The aim of our study was to identify the cross-reactive allergenic proteins from cashew and pistachio nuts. Methods: Extracted cashew and pistachio nut proteins were separated by means of 1- and 2-dimensional PAGE. Pooled human sera from cashew-allergic patients was tested... Show moreTitle: Allergenic cross-reactivity between cashew and pistachio nuts Rationale: Cashew and pistachio belong to Anacardiaceae family and strong allergenic cross-reactivity between nuts of these two species has been reported. The aim of our study was to identify the cross-reactive allergenic proteins from cashew and pistachio nuts. Methods: Extracted cashew and pistachio nut proteins were separated by means of 1- and 2-dimensional PAGE. Pooled human sera from cashew-allergic patients was tested for reactivity to soluble cashew and pistachio proteins by IgE immunoblotting after one-dimensional (1-D) and 2-D electrophoresis. The identities of the IgE-reactive bands from the pistachio immunoblot were further analyzed by means of N-terminal amino acid sequencing and comparison to previously published data from the cashew. ELISAs were performed using individual sera from subjects with cashew and tree nut allergy to assess the degree of IgE reactivity to cashew and pistachio nut extracts. Inhibition ELISA studies were conducted to assess the degree of allergenic cross-reactivity between cashew and pistachio nuts. Results: IgE immunoblots of cashew and pistachio extract probed with cashew-allergic sera identified proteins of 35kDa, 22kDa, and 7-9kDa. N-terminal amino acid sequencing of the IgE-reactive spots from pistachio immunoblot identified them as the acidic subunit, basic subunit of 11S globulin and 2S albumin seed storage proteins respectively. Seed storage proteins are known food allergens in cashew and have been designated as Ana o 1 (7S globulin), Ana o 2 (11S globulin) and Ana o 3 (2S albumin). ELISA results with ten individual cashew-allergic sera (two out of the ten patients have pistachio allergy, and the remaining eight patients had never eaten pistachio) showed IgE reactivity to both cashew and pistachio nut. Inhibition ELISA demonstrated that pre-incubation of sera with pistachio extract resulted in a marked decrease in IgE binding to cashew extract, and vice versa indicating allergenic cross-reactivity. Conclusion: The results demonstrate the presence of cross-reactive B cell epitopes on cashew and pistachio nut allergens. The plant taxonomic classification of cashew and pistachio nuts does appear to predict allergenic cross-reactivity. Show less

In male hamsters mating behavior is dependent on sufficient androgens and chemosensory input from the main olfactory and vomeronasal systems, whose central pathways contain cell bodies and fibers of gonadotropin-releasing hormone (GnRH) neurons. Regions of the medial amygdala (vomeronasal amygdala) contain androgen receptors and differentially process chemosignals with different social implications. According to published reports of "categorical" patterns of response, conspecific chemosensory... Show moreIn male hamsters mating behavior is dependent on sufficient androgens and chemosensory input from the main olfactory and vomeronasal systems, whose central pathways contain cell bodies and fibers of gonadotropin-releasing hormone (GnRH) neurons. Regions of the medial amygdala (vomeronasal amygdala) contain androgen receptors and differentially process chemosignals with different social implications. According to published reports of "categorical" patterns of response, conspecific chemosensory stimuli activate the anterior (MeA) and posterior (MeP) medial amygdala, while heterospecific stimuli only activate MeA, in male hamsters (and male mice). Furthermore, chemosignals with distinct social implications differentially activate the dorsal and ventral subregions of MeA and MeP (MeAd/v, MePd/v). In sexually-naïve male hamsters, lesions of the vomeronasal organ (VNX), but not the main olfactory bulb, impair mating behavior. Intracerebroventricular (icv)-GnRH restores mating in sexually-naïve VNX males and enhances medial amygdala (Me) activation by chemosensory stimulation. In sexually-experienced males, VNX does not impair mating and icv-GnRH suppresses Me activation. Thus, main olfactory input is sufficient for mating in experienced- but not naïve-VNX males. I tested whether GnRH enhances access of main olfactory input to the amygdala using icv-GnRH and either electrical or pharmacological stimulation of the main olfactory bulb (MOB), and then examined immediate early gene (IEG) expression there. Electrical stimulation of the MOB did not significantly activate the ipsilateral main olfactory cortex or amygdala in intact or VNX animals. When the IEG counts from both sides of the brain were averaged together, GnRH appeared to enhance activation in the medial amygdala in naïve-intact males, but appeared to decrease activation in naïve-VNX males. I concluded that electrical stimulation was not a sufficient means of driving main olfactory input to downstream brain regions, possibly due to activation of intra-bulbar inhibitory circuits. To alleviate this possible confound, I pharmacologically stimulated the MOB with a mixture of bicuculline methiodide and d,l Homocysteic acid. In sexually-naïve intact-males, MOB stimulation produced significant activation in MeAv and MePv. MePv activation is also characteristic of chemosensory stimuli from potential competitors and predators. In sexually-naïve VNX-males, in which GnRH facilitates mating, GnRH enhanced activation by MOB stimulation in posterodorsal medial amygdala (MePd), a region known to be rich in androgen resceptors and activated by conspecific reproductive chemosignals. Conversely, in sexually-experienced VNX-males, animals that do not require exogenous GnRH to mate normally after VNX, there is a depression in activation in MePd due to GnRH and stimulation in MePd, similar to its response to natural chemosensory stimulation. There also appeared to be a possible effect of VNX due to the difference in selective activation of GnRH in naïve-intact vs. naïve-VNX animals. MeP is also rich in steroid receptors and many chemosensory behaviors are steroid dependent. Therefore, I also tested the activation of androgen receptor (AR)-containing cells in Me after conspecific or heterospecific chemosensory stimulation. Conspecific and heterospecific chemosensory stimuli significantly activated AR-containing cells in Me and significantly increased the number of AR-positive cells in Me above control. The increase in the number of AR-ir cells produced by conspecific stimuli was also significantly above the numbers of AR-ir cells produced by the heterospecific stimulus. These effects may be due to increases of testosterone in response to chemosignals or circuit activity dependent on steroid levels. Future studies on castrated testosterone-replaced males will test these possibilities. The studies of this dissertation provide important information about the neurohormonal regulation of chemosensory and olfactory input to the medial amygdala. The integration of hormonal and chemosensory factors is vital to mating and other social behaviors, and thus species survival. The amygdala is crucial to this process in many vertebrate species, including the hamsters, which use chemicals to communicate with one another. This dissertation suggests, and provides some evidence for a part of the mechanism by which the amygdala accomplishes this integration. Show less

Date Issued

2009

Identifier

FSU_migr_etd-3669

Format

Thesis

Title

Analysis of IgE Reactivity to Pru Du 6, an 11S Globulin from Almond Nut, and Identification of Both Sequential and Conformational Epitopes.

Tree nuts are a widely consumed food and include walnut, cashew, almond, hazelnut, pistachio, pecan, chestnut, macadamia, and Brazil nut. Although enjoyed safely by most individuals, allergic reactions are common and ~0.6% of the US population is allergic to one or more tree nuts. An important IgE-reactive protein in almonds is prunin (Pru du 6), a legumin, which represents a major component of the seed and has been identified as an important allergen. Two prunin isoforms have been identified... Show moreTree nuts are a widely consumed food and include walnut, cashew, almond, hazelnut, pistachio, pecan, chestnut, macadamia, and Brazil nut. Although enjoyed safely by most individuals, allergic reactions are common and ~0.6% of the US population is allergic to one or more tree nuts. An important IgE-reactive protein in almonds is prunin (Pru du 6), a legumin, which represents a major component of the seed and has been identified as an important allergen. Two prunin isoforms have been identified: prunin 1 (Pru du 6.01) and prunin 2 (Pru du 6.02). IgE reactivity to recombinant Pru du 6.01 and Pru du 6.02 was found in the sera of 9 of 18 (50%) and 5 of 18 (28%) patients, respectively. To test for epitope stability, the recombinant proteins (rPru du 6) were treated with various denaturants. Immunodot blotting assays revealed the presence of both stable (sequential) and unstable (conformational) rPru du 6 epitopes. The sequential IgE binding epitopes on Pru du 6 were identified by solid-phase overlapping peptide analysis (SPOTs assay). Six IgE-binding sequential epitope-bearing peptides were found on Pru du 6.01 and eight on Pru du 6.02 using almond-allergic sera. Murine anti-almond IgG mAbs also showed greater reactivity to rPru du 6.01 than to rPru du 6.02. The sequential epitopes targeted for several mAbs (4G2, aa 353LQQERQQ359 ; 2B4 and 5D1, aa 120QQGRQQ125) were identified by SPOTs assay. The 5D1 and 2B4 epitopes were found to directly overlap with an IgE binding epitope on Pru du 6.01. Immunoblots, immunodot blots, and inhibition immunoblots indicate that Pru du 6.01 is the predominate isoform in the nut. To investigate conformational epitopes on prunin (Pru du 6), phage display technology was used to identify IgE-binding epitopes. Total IgE was purified from an almond-allergic patient by affinity chromatography and used to capture phage displaying 12 aa-long peptides that mimic prunin epitopes. The phage sequences were analyzed using the Pepitope program and three conformational epitopes were identified (epitope 1, E49,N78, L80, L82, P83, L244, A245, N290, R312, L314, G316, N322, I325, Q326; epitope 2, V104, F105, H190, Q191, T193, P205, A206, G207, V208, V327, R328, G329, N330, L331, D332, F333; epitope 3, H227,S229, S230, D231, H232, F411, W431, V433, N434, H436, V451, Q473, N474, H475, G476, T493, N496, A497, F498, L502). One of these epitopes partially overlaps a sequential epitope previously identified by the SPOTs assay. Conformational epitope mapping studies were also performed using a murine monoclonal antibody (mAb) 4C10. This mAb reacts exclusively with non-reduced native prunin in immunoblotting assays, indicating that 4C10 binds to a conformational epitope expressed on prunin that is dependent on the large and small subunit association. Inhibition ELISA assays found that human IgE and IgG binding epitopes (sequential and/or conformational) overlap or sterically hinder 4C10 binding to prunin. To identify the epitope, hydrogen/deuterium exchange (HDX) monitored by 14.5 T Fourier transform ion cyclotron resonance mass spectrometry was performed on prunin and the 4C10-prunin complex. Comparison of deuterium uptake between the free vs. mAb-bound prunin identified several epitope candidate peptides that differ in deuterium uptake, suggesting that these peptides are part of the 4C10 epitope. Analyses of chimeric molecules using the homologous soybean allergen, Gly m 6, and alanine mutants further localized the epitope to three discontinuous strands (aa 21-45, 320-328, and 460-465). These data demonstrates that HDX-MS is a useful technique to aid in the identification of unknown conformational epitopes on native tree nut allergens. Show less

The nest architecture of underground ant nests was studied in Formica pallidefulva. These ants build shallow (30-45 cm deep) nests, which consisted of more or less vertical shafts that bear chambers. Shafts appeared to be modular units of nest growth; nests were enlarged by adding more shafts or extending previously existing ones. The nests were top-heavy, their volume declining exponentially with depth. The total volume of the nest was strongly correlated with the number of worker occupying... Show moreThe nest architecture of underground ant nests was studied in Formica pallidefulva. These ants build shallow (30-45 cm deep) nests, which consisted of more or less vertical shafts that bear chambers. Shafts appeared to be modular units of nest growth; nests were enlarged by adding more shafts or extending previously existing ones. The nests were top-heavy, their volume declining exponentially with depth. The total volume of the nest was strongly correlated with the number of worker occupying the nest. Several rules and templates that may be used by workers for nest construction were determined: (a) chambers are formed in the direction of the tunnels leading up to them, (b) the amount of soil excavated per unit time was related to the soil temperature and the moisture content of soil. The amount of time and energy required to construct a typical nest were estimated using digging ability parameters estimated in the lab. It was found that if a colony was to move twice a year, it would expend 21% of its energy intake and 6% of its worker time on nest excavation. Show less

Date Issued

2002

Identifier

FSU_migr_etd-2444

Format

Thesis

Title

Assessment of Mercury (II) Species Bioavailability Using a Bioluminescent Bacterial Biosensor.

The objective of this research was to investigate the bioavailability of various Hg(II) species in laboratory and natural solutions using a bioluminescent bacterial biosensor. The Hg(II) bacterial biosensor is a genetically engineered E. coli strain, which produces firefly luciferase in proportion to its exposure to bioavailable Hg(II). The amount of luciferase inside the cells is assessed through its bioluminescent reaction with luciferin. A new analytical protocol for the use of bacterial... Show moreThe objective of this research was to investigate the bioavailability of various Hg(II) species in laboratory and natural solutions using a bioluminescent bacterial biosensor. The Hg(II) bacterial biosensor is a genetically engineered E. coli strain, which produces firefly luciferase in proportion to its exposure to bioavailable Hg(II). The amount of luciferase inside the cells is assessed through its bioluminescent reaction with luciferin. A new analytical protocol for the use of bacterial bioassays to study the bioavailability of trace elements and relate it to their modeled chemical speciation was developed. The biosensor showed high specificity for Hg(II) and good reproducibility. The detection limit of the method (0.7 pM) is superior to most of the other reported Hg(II) biosensors and adequate to analyze mercury bioavailability at natural levels. The feasibility of using this new biosensor method to analyze natural samples was assessed. An approach for estimating conditional equilibrium constants for the complexation of Hg(II) by natural ligands is presented. The influence of inorganic and organic ligands on the Hg(II) speciation and bioavailability was investigated. Chloride titration results suggested that neutral HgCl20 and Hg(OH)20 complexes were more bioavailable than anionic Hg(II) chloride complexes. The addition of EDTA decreased the biosensor's response in both synthetic solutions and natural samples. This reduction was proportional to the total EDTA concentration. Kinetic experiments were performed to evaluate the Hg(II) uptake process by the bacterial biosensor. The experimental data agreed with the ultrasensitive kinetic model proposed for the Mer R protein response to Hg(II). The half-saturation constant for the enzymatic reaction was estimated. The Hg biosensor response displayed non-linear increases to both increasing exposure times and Hg(II) concentrations. According to the kinetic results, both non-linear behaviors are probably due to the saturation of the mer operon/Mer R protein with Hg(II). The analysis of the biosensor cells for their total Hg(II) concentration during a kinetic assay revealed no evidence for saturation of the Hg(II) uptake process. The kinetic results agree with the titration data and support the hypothesis that the diffusion of neutral Hg(II) complexes is an important Hg(II) uptake mechanism for bacterial cells. Show less

Date Issued

2004

Identifier

FSU_migr_etd-1114

Format

Thesis

Title

The Association of Pectobacterium, a Plant Pathogen, with the Carapace of the Caribbean Spiny Lobster, Panulirus Argus.

The Caribbean spiny lobster, Panulirus argus, is found throughout the Florida Keys and Dry Tortugas. In 1998, lobsters were discovered with small black, necrotic lesions on the carapace some associated with trauma. Since then the number of occurrences of shell disease has increased, a potential problem for the lobster fishery in the southeast. In an effort to determine the etiology of shell disease, bacterial samples were obtained from healthy and diseased lobsters from 28 different locations... Show moreThe Caribbean spiny lobster, Panulirus argus, is found throughout the Florida Keys and Dry Tortugas. In 1998, lobsters were discovered with small black, necrotic lesions on the carapace some associated with trauma. Since then the number of occurrences of shell disease has increased, a potential problem for the lobster fishery in the southeast. In an effort to determine the etiology of shell disease, bacterial samples were obtained from healthy and diseased lobsters from 28 different locations in the Florida Keys and Dry Tortugas. DNA fingerprinting of the 16s-23s rRNA intergene region (IGR) placed 487 isolates into five major groups of gamma-Proteobacteria, one group of Gram-positive organisms, and individuals of low occurrence were placed in an "Other" group. Sequencing of the 16s rRNA gene and GenBank BLAST analysis identified the genus Erwinia was the second largest group that consisted of 89 isolates. Out of the 89 isolates, 88 were identified as Erwinia cypripedii. The E. cypripedii sequences were aligned with sequences from known strains of marine bacteria; members of the Erwinia, Brenneria, Pectobacterium, Pantoea genera; and other members from the gamma-Proteobacteria group. After performing phylogenetic analyses using the program PAUP*, the lobster isolates were placed into a monophyletic group distinct from, but closely related to, E. cypripedii. Erwinia spp. are normally associated with plants and rotting vegetation, and therefore, not seen in a marine environment especially on the carapace of an invertebrate. Since the isolates showed little diversity by 16S rRNA analysis, two protein coding genes, recombinase A (recA) and recombination-dependent growth C (rdgC), were picked to further analyze the Erwinia isolates. Once again the recA and rdgC gene sequences showed very little diversity among the isolates. Each isolate was also screened for extracellular enzyme production to see if they could break down proteins, lipids, and/or chitin, to establish a possible pathogenic role. Show less

Non-random mating is presumed to be an important mechanism that allows for the maintenance of genetic variation. Assortative mating has been studied extensively in organisms that possess defined ways in which sperm is transferred to eggs (e.g. via copulation, courtship or vector assisted pollination in plants), but rarely in broadcast spawners. Broadcast spawning is perceived as a mating event that allows for mixing of gametes and promotes random mating. However, there are multiple pathways... Show moreNon-random mating is presumed to be an important mechanism that allows for the maintenance of genetic variation. Assortative mating has been studied extensively in organisms that possess defined ways in which sperm is transferred to eggs (e.g. via copulation, courtship or vector assisted pollination in plants), but rarely in broadcast spawners. Broadcast spawning is perceived as a mating event that allows for mixing of gametes and promotes random mating. However, there are multiple pathways in which spawning adults can affect fertilization of gametes in non-random ways. For example, positive assortative mating can occur in broadcast spawners if similar phenotypes spawn closer together in space or time, or possess similar gamete recognition proteins that expedite fertilization. Here, I propose to examine assortative fertilization, patterns of aggregation and gamete recognition protein genotype of the sperm bindin gene as a function of spine color in the sea urchin Lytechinus variegatus as well as evaluating deviations from Hardy-Weinberg Equilibrium (HWE) based on color. Results indicate that laboratory crosses of urchins within color morphs yielded higher fertilization success than crosses between color morphs. Field surveys determined that these sea urchins are aggregating by color at times of their reproductive season when they are more likely to spawn. Tests for HWE using field data of urchin phenotypes suggest strong deviations from HWE. However, DNA sequences of regions of the sperm bindin gene for sea urchins of different color do not show evidence of genetic structure of the population. Paternal success in broadcast spawners is largely determined by the proximity of males to spawning females and the compatibility between them at the time they release their gametes. Selection is predicted to favor traits and behaviors that increase the likelihood of spawning near a more compatible neighbor. These results provide strong evidence for assortative mating and an explanation for the maintenance of color variation in this species. Show less

Date Issued

2017

Identifier

FSU_SUMMER2017_Moscoso_fsu_0071N_14093

Format

Thesis

Title

The Batoid Tree of Life: Recovering the Patterns and Timing of the Evolution of Skates, Rays and Allies (Chondrichthyes: Batoidea).

Batoid fishes (skates, stingrays and allies) comprise the majority of species diversity and morphological disparity among chondrichthyans, one of the two primary divisions of extant jawed vertebrates. The largely recent and growing interest in batoid evolution has emphasized the need for a well-supported phylogeny against which evolutionary changes in traits can be interpreted. While batoids are morphologically well characterized and have an excellent fossil record, there is currently no... Show moreBatoid fishes (skates, stingrays and allies) comprise the majority of species diversity and morphological disparity among chondrichthyans, one of the two primary divisions of extant jawed vertebrates. The largely recent and growing interest in batoid evolution has emphasized the need for a well-supported phylogeny against which evolutionary changes in traits can be interpreted. While batoids are morphologically well characterized and have an excellent fossil record, there is currently no consensus on the interrelationships of family-level taxa. Patterns of evolution within the two largest groups of batoids, skates and stingrays, also remain obscure. This dissertation presents novel frameworks for interpreting the patterns and timing of batoid evolution based on molecular data, morphology, and fossils. I recovered a resolved and time-calibrated phylogeny of the major extant groups of batoids using mitochondrial genomes, two independent nuclear markers, and fossil ages. Taxon sampling included 37 ingroup species from 22 of 23 families. Data partitioning schemes, potential biases in the sequence data, the relative informativeness of each fossil, and ancestral state reconstructions were explored. The molecular data set was then expanded with additional species in order to address questions at a finer taxonomic scale, in particular among skates and stingrays. Two nuclear and two mitochondrial markers were sequenced for 87 batoid species across 52 of 81 genera. A similar analytical approach was applied to this larger data set. I also performed a morphology-based phylogenetic analysis in order to interpret accurately coded morphological characters against the molecular frameworks. I updated the data set of McEachran and Aschliman (2004) with a number of corrections and modifications, and added new characters from the synarcual and other chondroskeletal structures. The molecular phylogenies indicate that the major lineages of batoids originated in relatively rapid sequence, followed by long periods of independent evolution. These trees corroborate morphology-based hypotheses in many respects, but have strongly divergent implications in others. Lineages inferred to be distantly related, such as skates and stingrays, or sawfishes and sawsharks, are indicated to have achieved very similar, specialized body plans through convergence. Skates and stingrays are unique among batoids in exhibiting a highly depressed disc supported to the apex by fin rays, and swim by passing waves along the lateral margin of the pectoral fin without additional propulsion by lateral motion of the tail and caudal fin. The plesiomorphic mode of locomotion and body plan for most batoid groups was probably not shark-like, as in sawfishes. Instead, it is inferred to be a combination of pectoral fin undulation and shark-like axial locomotion, which is correlated with a broader pectoral disc and reduced tail compared to the sawfish body plan. The higher-resolution molecular phylogeny is in many cases congruent with previous morphological studies or biogeography. Exceptions typically suggested a potential weakness in the molecular data, suggested that a morphology-based classification scheme is likely based on convergent characters or ignores a highly divergent morphotype nested within otherwise similar taxa, or helped resolve taxonomic confusion based on informal re-assignment by authority. Potentially non-monophyletic families and genera were identified for future study with expanded taxon sampling, additional sequence data and morphological re-evaluation. The origin of Batoidea is estimated to have occurred in the Late Triassic, with the major groups diverging throughout the Jurassic and possibly into the Cretaceous. Radiations of each major crown group are indicated to have occurred from the Late Cretaceous to the Cenozoic. The tree shape recovered for all major batoid groups, with long internal branches subtending subsequent radiations around the Cretaceous/Tertiary boundary, suggests that batoid standing diversity may be due in large part to lineage pruning and/or rapid radiation into vacated niche space. This is consistent with the fossil record, which suggests that batoids were more severely affected by the end-Cretaceous extinction event than were the other neoselachians. The updated morphological phylogeny included several key changes from earlier hypotheses and more closely approximates the molecular frameworks. There remain conflicts between morphological and molecular trees, such as the phylogenetic placements of skates and thornbacks, which currently appear to be difficult to reconcile. Future attempts to reconcile molecules and morphology will require expanded data sets and investigation of potential sources of error in each. Show less

In many animal species there is pronounced and repeatable variation among individuals in their expression of suites of behaviors, resulting in consistent behavioral types. These behavioral types are characterized by measures like the degree of boldness, aggression, activity, and exploratory behavior. While the prevalence of behavioral types and their importance in terms of natural selection have become apparent in recent years, their importance in determining the outcome of sexual selection... Show moreIn many animal species there is pronounced and repeatable variation among individuals in their expression of suites of behaviors, resulting in consistent behavioral types. These behavioral types are characterized by measures like the degree of boldness, aggression, activity, and exploratory behavior. While the prevalence of behavioral types and their importance in terms of natural selection have become apparent in recent years, their importance in determining the outcome of sexual selection remains imperfectly understood. In this dissertation I examined the role of behavioral types in sexual selection in the bluefin killifish, Lucania goodei, as well as the influence of environmental and genetic factors on the expression of behavioral types. In chapter two I investigated how female choice and male competition interact in the bluefin killifish in a 3-staged experiment where (1) females could choose between two males, (2) those males could interact in the presence of that female, and (3) females and males could freely interact and spawn. In the pairwise stages (1 and 2), females displayed pronounced preferences between males and male competition produced a distinctly dominant individual. None of the morphological traits, including color, measured in males were associated with either female preference or male dominance. When all three fish interacted (stage 3), male mating activity level (behavioral type) was the sole predictor of spawning success. Males with elevated activity levels were more aggressive toward males and females, exhibited intensified courtship, and obtained more spawns. Female preference did not predict the number of spawns with a male but it did predict her latency to spawn; females spawned more quickly with preferred males. Thus, male competition and female choice interact to determine reproductive success but there is evidence for conflict and a cost to females of associating with dominant males. Reproductive success in this species is not easily predicted from simple measures of morphology or female preference, and is influenced by complex social interactions, both between males, and between males and females. The outcomes of these social interactions are in turn influenced by the behavioral types of the participants. In chapter three I examined whether repeatable personality differences among males are associated with repeatable outcomes of male-male interactions within the mating context. Specifically I examined the repeatability of individual differences in mating behaviors and the stability of dominance status, which partially determines mating success in the bluefin killifish. The expression of male behaviors in competition between males and female courtship was significantly repeatable over a five-week time period; the number of aggressive behaviors to males, to females, and the number of courtship bouts had significant repeatabilities of 0.71, 0.72, and 0.65 respectively. A male's behavioral type within the mating context, as measured by a composite measure of the overall level of mating behavior activity, was significantly repeatable at 0.75. Males showed repeatable, linear dominance hierarchies and a male's rank in the hierarchy was highly correlated with his behavioral type. Neither behavioral type nor dominance status was associated with body size or body condition. The repeatability of behavioral types and stability in the outcomes of aggressive interactions, suggest that these behavioral types are inherent characteristics of individuals rather than short-term responses to recent social experience or daily levels of food or stress. In chapter four, I examined how differences between individuals in behavioral type arise. Specifically I examined whether nutritional and social conditions experienced by individuals early in life affect the development of behavioral type and behavioral expression later in life in the bluefin killifish. As fry, individuals experienced either high food or low food levels and within each food treatment, individuals experienced one of three social environments: they were reared with one adult male, one adult female, or no adult, producing six treatment combinations. Social treatments were removed at 5 months and food manipulations were ceased at 6 months, after which individuals were fed ad libitum until behavioral testing at 10 months. Males reared on high food and low food were paired with one another within a social treatment and tested in a female choice trial and male dominance trial. Food and social treatments produced strong, synergistic effects on growth; individuals raised at high food were larger than those raised at low food and high food individuals reared with an adult female grew faster than those in all other treatments. There was no evidence for compensatory growth after release from treatments imparting slow growth. Despite the effects of early experience on growth and body size, I was unable to detect any comparable effect on behavioral variation. Male behavioral type and behavioral expression were not significantly affected by food level and this did not vary across social treatments. High food and low food males did not differ significantly in their likelihood of becoming dominant as adults or of being preferred by females. These results suggest that an individual's early rearing environment, at least the particular aspects manipulated here, have little influence on the development of behavioral type in this species. Given that personalities can affect fitness, understanding the environmental and genetic factors that influence personalities, as well as the associations among behaviors across different contexts are crucial to understanding the evolutionary response of personality to direct and indirect selection. In chapter five, I performed a series of paternal half-sib crosses to estimate the heritability of a number of distinct behaviors within and across three different contexts (mating, response to a predator, recovery from stress) as well as whether there are phenotypic and genetic correlations among behaviors exhibited within and across contexts. Despite the presence of strong phenotypic correlations among the behaviors exhibited in the mating context, my results indicate that only courtship behaviors appear to be significantly heritable and there is little evidence for significant additive genetic variance in aggressive behaviors. In addition, I found no evidence for phenotypic correlations among traits expressed outside the mating context, nor evidence for additive genetic variation for those behavioral traits. The results of my dissertation point to more complex sources of individual variation in personality and, concomitantly, more complex patterns of selection. I found that while variation in three behaviors formed an integrated personality, in only one behavior was that variation based on substantial heritable genetic variation. What emerges as individual personality could be the result of individual variation in a single behavior and the network of provocation and response that is built by social interactions. This complexity opens new avenues for future empirical work and new possibilities for understanding the subtle interplay of genetic and environmental foundations for the behaviors seen in complex social systems. Show less

Date Issued

2009

Identifier

FSU_migr_etd-2560

Format

Thesis

Title

Biomechanics and the Ontogeny of Feeding in the American Alligator (Alligator Mississippiensis): Reconciling Factors Contributing to Intraspecific Niche Differentiation in a Large Bodied Vertebrate.

As vertebrates attain a larger body size, the relative growth of various body parts results in differential rates of growth across the organism and may result in substantial impacts on mechanical performance. To deal with this problem, shape changes often accompany somatic growth, and the nature of these changes is thought to reflect a tight relationship between the morphological form of an organism's anatomy and its function within a given environment. In this context, the feeding anatomy of... Show moreAs vertebrates attain a larger body size, the relative growth of various body parts results in differential rates of growth across the organism and may result in substantial impacts on mechanical performance. To deal with this problem, shape changes often accompany somatic growth, and the nature of these changes is thought to reflect a tight relationship between the morphological form of an organism's anatomy and its function within a given environment. In this context, the feeding anatomy of vertebrates is a consummate form-function relationship. An extreme example of a taxon that undergoes ontogenetic dietary shifts is the American alligator, Alligator mississippiensis, which traverses a 5000-fold increase in mass during its lifetime. As a consequence, this species must cope with changes in its feeding functional morphology (i.e., size and shape of dental and musculoskeletal attributes) while exploiting varying ecological feeding niches. Hatchlings start out as insectivorous neonates but abandon that feeding niche as larger body size allows them access to a wider range of prey items, first frogs and fish and other small, compliant prey before finally reaching the adult ecomorphology where they access more robust quarry. Absolute body size and bite-force positive allometry play an important role in these transitions. Therefore, to understand the nature of the anatomical changes that underpin these factors and thus facilitate dietary niche transitions, I dissected a growth series of wild A. mississippiensis. I standardized the topology, attachment points, and naming scheme for the jaw adductor musculature and quantified the growth of the cranial skeleton, jaw adductor muscle system, and dental form throughout ontogeny. I derived mathematical models of bite-force and hold-force generation based on these data, and tested them against additional developmental series of known bite-force A. mississippiensis. I developed and implemented a novel technique to identify the aerobic capacity of the jaw adductor muscles, called Muscle Oxidative Inference Analysis (MOIA). Finally, dental pressures were quantified using bite forces and dental morphology. These data demonstrate that after hatching, larger body size (25 cm snout-vent length, SVL) initially allows A. mississippiensis access to increasingly larger, compliant prey. At 45 cm SVL positive allometry in the post-orbital growth of the skull and M. Pterygoideus ventralis muscles in addition to substantial changes in the aerobic capacity of some jaw adductor muscles facilitates access elusive terrestrial prey such as birds and small mammals. By 75 cm SVL, subtle changes in dental form along with bite-force positive allometry make it possible for this taxon to generate tooth pressures that can fracture and mechanically fail the bony carapaces of turtles. Finally, large adult body size (150+ cm SVL) and continued positive allometry in the musculoskeletal attributes of its jaw adductor system and further augmented oxidative capacity of its adductor muscles allow A. mississippiensis to secure large game mammals such as deer, boar, and cattle. Show less

Date Issued

2010

Identifier

FSU_migr_etd-7143

Format

Thesis

Title

Bite-Force Generation and Feeding Biomechanics in the Loggerhead Musk Turtle, Sternotherus Minor: Implications for the Ontogeny of Performance.

Ontogenetic growth can profoundly affect the ability of organisms to perform ecologically-relevant feeding tasks that ultimately impact survival. In particular, bite-force generation is exceedingly important for vertebrates that process and consume robust prey (i.e. durophagy). Consequently, bite-force generation used in durophagy is a suitable parameter to investigate the functional relationships between musculoskeletal biomechanics, feeding performance, and ecology. I studied the ontogeny... Show moreOntogenetic growth can profoundly affect the ability of organisms to perform ecologically-relevant feeding tasks that ultimately impact survival. In particular, bite-force generation is exceedingly important for vertebrates that process and consume robust prey (i.e. durophagy). Consequently, bite-force generation used in durophagy is a suitable parameter to investigate the functional relationships between musculoskeletal biomechanics, feeding performance, and ecology. I studied the ontogeny of bite-force generation and feeding biomechanics in the durophagous turtle, Sternotherus minor. Across an ontogenetic series of 75 S. minor, craniofacial growth was characteristized by allometric increases (i.e. positive allometry) in the dimensions of the head and beak. Moreover, bite-force generation increased with positive allometry relative to body and head dimensions. These results indicate that ontogenetic modifications to the lever mechanics of the jaw system, and/or changes in the size (i.e. mass) and/or physiology (e.g., fiber lengths, degree of pennation) of the jaw adductor musculature have more explanatory power for bite-force generation than external measures. A detailed, quantitative examination of the musculoskeletal biomechanics was performed to elucidate how these animals are capable of generating disproportionately high bite forces throughout ontogeny. Mechanical levers, muscle masses, and muscle architecture (fiber lengths and pennation angles) were measured from an ontogenetic series of 30 S. minor. With these data, a biomechanical model of the feeding apparatus was developed that accurately predicts individual and ontogenetic scaling of bite forces. Increasing muscle masses and changing the muscle architecture resulted in an increase in total physiological cross-sectional area (PCSA) of the jaw adductor muscles that was proportional to changes in bite-force generation. These results indicate that the disproportionate increase in bite-force generation relative to skull length found in S. minor is explained by allometric changes in muscle size and architecture that collectively act to allometrically elevate the PCSA and muscle force. The alternative explanation of improving the mechanical leverage was not supported. Dietary data indicated that the positive allometry in bite-force generation observed in S. minor is tightly linked to the incorporation of exponentially larger snails into the diet and positive allometry of the forces required to fracture the largest dietary items. These forces were found to be greater than the observed and theoretical bite forces, which suggested that fatigue failure resulting from multiple bite-force loadings may allow S. minor to fracture snails at lower compressive forces and access large snails that are apparently outside the range of their bite-force capacity. Moreover, age-based growth patterns for bite-force generation fit a logistic growth curve and reveal a close relationship with the forces required to fracture snails found in the diet. The results of this study provide empirical evidence that ontogenetic changes to musculoskeletal morphology and ecology are inextricably linked by the performance of the feeding apparatus in S. minor. Such results are exceedingly important for establishing a baseline from which to explore the mechanistic and functional issues that underlie the evolution of phenotypic traits among vertebrates. Show less

Voltage-gated potassium ion channels such as Kv1.3 have a role in altering excitability of neurons. The neuron has to have a sophisticated mechanism to regulate the modulation, expression, turnover and distribution of ion channels. Ion channels, like Kv1.3, become crucial in affording the neuron one of a symphony of players that can strategically play their part in transmitting electrical and chemical signals into meaning. This dissertation uses electrophysiology and biochemistry to... Show moreVoltage-gated potassium ion channels such as Kv1.3 have a role in altering excitability of neurons. The neuron has to have a sophisticated mechanism to regulate the modulation, expression, turnover and distribution of ion channels. Ion channels, like Kv1.3, become crucial in affording the neuron one of a symphony of players that can strategically play their part in transmitting electrical and chemical signals into meaning. This dissertation uses electrophysiology and biochemistry to investigate how brain-derived neurotrophic factor (BDNF) and TrkB utilize a very simple aspect of the biochemistry of Kv1.3 to specifically modulate the biophysical properties, expression and turnover of Kv1.3. Acute BDNF application suppresses Kv1.3 current and results in phosphorylation of tyrosine residues 111-113, 137 and 449. There is a delicate balance of other downstream cellular components; N-Shc, Grb10 and PSD95 that disrupt this BDNF induced current suppression. N-Shc disrupts a post-phosphorylation event that usually leads to BDNF-evoked Kv1.3 current suppression, and N-Shc causes phosphorylated Kv1.3 to be retained in the membrane. Grb10 and PSD95 left-shift the voltage at half-activation of Kv1.3 and this effect may not be phosphorylation-dependent. Grb10 also reduces Kv1.3 expression and causes redistribution of membrane inserted Kv1.3. The presence of BDNF activated tropomyosin-related kinase B (TrkB) increases the phosphorylation of Kv1.3 tyrosine residues and increases Kv1.3 expression by two fold. TrkB also increases the half-life of Kv1.3 and this can account for the increase in protein expression. Kv1.5 is another member of the Shaker family but TrkB decreases the expression of Kv1.5. The insulin receptor (IR) is also a tyrosine kinase like TrkB however, IR decreases Kv1.3 expression and has no effect on Kv1.5 expression. These effects demonstrate that the TrkB and IR mediated regulation of Kv1.3 expression is not a promiscuous interaction of Shaker channels with receptor tyrosine kinases. Given the prominence of Kv1.3 in the olfactory bulb, one can hypothesize that the above interactions can play a part in modulating the function of Kv1.3 during development, learning and injury in the olfactory bulb. The neuron can utilize these interactions to regulate Kv1.3 and change how Kv1.3 contributes to shaping the neuron's response. Show less

A population of Odontomachus brunneus, a primitive species of Ponerine ants, located in north Florida was studied for a one-year period. Through nest excavation and colony census, the annual cycle of reproduction and colony growth was determined with nests exiting a period of winter inactivity in late April and beginning brood production. Early brood is a combination of both sexuals and workers, with sexuals present in nests during June and July. After production of sexuals in May and June,... Show moreA population of Odontomachus brunneus, a primitive species of Ponerine ants, located in north Florida was studied for a one-year period. Through nest excavation and colony census, the annual cycle of reproduction and colony growth was determined with nests exiting a period of winter inactivity in late April and beginning brood production. Early brood is a combination of both sexuals and workers, with sexuals present in nests during June and July. After production of sexuals in May and June, all subsequent brood produced were found to be workers through dissection of pupal cocoons. Brood production ceased in October, with the final pupae eclosing in November at which time the colonies began a four month period of relative inactivity. Within-nest seasonal energy allocation was determined by fat extraction. Seasonal energy stores coincided with the annual cycle of reproduction, with workers declining in energy stores (fat) during initial brood production and regaining these stores after production of brood in preparation for the winter season during which colonies are primarily inactive. Comparison of body fat provided the relative ages of worker ants, which suggested that O. brunneus nests display internal age stratification throughout the majority of the year with older, leaner workers being found in the upper chambers of most nests and younger, fatter workers in the lower chambers. Using a combination of mark-recapture of foragers and nest excavation, the proportion of foragers per colony was shown to include a mean of 77% (S.D. 22) of the workforce. This proportion was not related to colony size. Female alates were also found to be a part of the foraging population of colonies. Show less

Date Issued

2009

Identifier

FSU_migr_etd-4225

Format

Thesis

Title

Broad, an Active Participant in Drosophila Oogenesis with Broad Functions.

The follicular epithelium (FE) of the Drosophila egg chamber is an excellent model system to study cell-cycle regulation, cell differentiation and cell migration in development. During oogenesis, follicle cells sequentially undergo three distinct cell-cycle programs: the mitotic cycle (stage 1-6), endocycle (stage 7-10a), and gene amplification (stage 10b-13). Notch signaling plays a central role in regulating follicle-cell differentiation and cell-cycle switches; its activation and... Show moreThe follicular epithelium (FE) of the Drosophila egg chamber is an excellent model system to study cell-cycle regulation, cell differentiation and cell migration in development. During oogenesis, follicle cells sequentially undergo three distinct cell-cycle programs: the mitotic cycle (stage 1-6), endocycle (stage 7-10a), and gene amplification (stage 10b-13). Notch signaling plays a central role in regulating follicle-cell differentiation and cell-cycle switches; its activation and inactivation in follicle cells are essential for the mitotic cycle/endocycle (M/E) and the endocycle/gene amplification (E/A) switches, respectively. In my dissertation, I mainly focus on Notch signaling and its downstream target broad (br). In the first part of the dissertation, I introduce the background information of the egg chamber system, Notch signaling and other associated factors. In the second part, I describe a screen strategy to identify novel genes involved in Notch-mediated follicle cell differentiation and cell cycle switches. In the third part, I select a Notch target gene br from the above-mentioned screen and study its regulation and functions. I will show br, encoding a small group of zinc-finger transcription factors resulting from alternative splicing, is a transcriptional target of Notch nuclear effector Suppressor of Hairless (Su(H)). The early pattern of Br in the FE, uniformly expressed except in the polar cells, is established by Notch signaling around stage 6, through the binding of Su(H) to the br early enhancer (brE) region. My findings also suggest an important role of br in the timing of follicle cell development during the M/E switch. In the fourth part, I report the uniform pattern of Br in the follicular epithelium is gradually lost in the anterior follicle cells (stretched cells and border cells) from stage 9 to 10a during oogenesis. This downregulation of Br is functionally significant for proper stretched-cell stretching. I also find ecdysone and JAK/STAT signaling mediate the downregulation of Notch-maintained Br. Together, My research investigates the complex Notch signaling network, and reveal that Notch-directly-regulated Br interacts with the ecdysone and JAK/STAT pathways, serving as an important spatiotemporal cue for proper cell differentiation and morphogenetic movement during Drosophila oogenesis. Show less

The cardiac thin filament is a highly ordered regulatory system for cardiac contraction. Regulatory proteins, a-tropomyosin (a-Tm) and troponin (cTn, composed of cTnT, cTnI, and cTnC), provide a Ca2+-dependent mechanism for controlling actin-myosin interactions that underlie cardiac muscle contraction and relaxation. These regulatory proteins may be altered or modified in a variety of ways in vivo, e.g. by mutation or post-translational modifications, which can alter the function and possibly... Show moreThe cardiac thin filament is a highly ordered regulatory system for cardiac contraction. Regulatory proteins, a-tropomyosin (a-Tm) and troponin (cTn, composed of cTnT, cTnI, and cTnC), provide a Ca2+-dependent mechanism for controlling actin-myosin interactions that underlie cardiac muscle contraction and relaxation. These regulatory proteins may be altered or modified in a variety of ways in vivo, e.g. by mutation or post-translational modifications, which can alter the function and possibly the structure of the whole heart over time scales both short (beat-to-beat) and long (years). Familial Hypertrophic Cardiomyopathy (FHC) is a disease caused by genetic mutations in sarcomeric genes, notably cTnI and a-Tm, that result in left and/or right ventricular hypertrophy. Three cTnI FHC mutations (P82S, D196N, and S199N) are characterized in Chapter 1 in relation to the modulation of their respective Ca2+-sensitivities of individual actin filaments in the in vitro motility assay. One a-Tm FHC mutant (E180G) that causes an increase in Ca2+-sensitivity in the in vitro motility assay is further studied in Chapter 2. Chapter 3 characterizes a commercially available cTn sample from Research Diagnostics for post-translational modifications. Show less

Individual members of many species use chemical signals detected by the vomeronasal system to communicate with other members of the same species. Over the past two decades, research in the chemical senses field has focused on peripheral detection and processing of chemosensory signals by the vomeronasal organ (VNO). Not as much focus has been placed on central processing of these signals once they are detected and communicated to the accessory olfactory bulb (AOB). The studies included in... Show moreIndividual members of many species use chemical signals detected by the vomeronasal system to communicate with other members of the same species. Over the past two decades, research in the chemical senses field has focused on peripheral detection and processing of chemosensory signals by the vomeronasal organ (VNO). Not as much focus has been placed on central processing of these signals once they are detected and communicated to the accessory olfactory bulb (AOB). The studies included in this dissertation were designed to investigate neuronal activation in the medial amygdala, an area of the brain that gets direct input from the accessory olfactory bulb (AOB). Based on immediate early gene (IEG) expression in activated neurons, I found that the medial amygdala responded differently to pheromonal chemosignals from animals that were conspecific (same species) versus heterospecific (different species). The anterior medial amygdala (MeA) responded to both, but posterior medial amygdala (MeP) responded only to conspecific stimuli and was suppressed during responses to heterospecific stimuli or artificial (non-biological) stimuli. These data provide the first report of categorical discrimination of chemosensory signals in the medial amygdala. This categorization was not apparent in the AOB so it appears to reflect a second level of sensory analysis. Additional studies indicated a reciprocal relationship between activation in MeP and in adjacent inhibitory intercalated nucleus (ICN) cells. MeP is the brain area that only responded to conspecific stimuli. Its lack of activation in MeP with heterospecific stimuli is accompanied by selective suppression in MeP neurons expressing GABA-a Receptor and occurs concurrently with significant activation in GABA immunoreactive cells of the adjacent ICN. The ability of neurons in medial amygdala to show a discrimination between conspecific and heterospecific stimuli was not dependent on main olfactory input and MeA is the first place in the vomeronasal pathway where all the information from rostral and caudal accessory olfactory bulb comes together. Immediate early gene expression does not reveal all the neural activity of the brain, but within the limits of this method all indications are that discrimination occurs in the anterior medial amygdala. Show less

The time at which DNA replicates during S-phase (replication timing; RT) is a precisely orchestrated, yet large-scale epigenetic property that offers an unparalleled window into the structure and regulation of the genome. While the timing program has been studied in many contexts and its significance is now well-established, the mechanisms that establish it have proven elusive, and recent studies have shed light on temporal and spatial aspects to replication control that were previously... Show moreThe time at which DNA replicates during S-phase (replication timing; RT) is a precisely orchestrated, yet large-scale epigenetic property that offers an unparalleled window into the structure and regulation of the genome. While the timing program has been studied in many contexts and its significance is now well-established, the mechanisms that establish it have proven elusive, and recent studies have shed light on temporal and spatial aspects to replication control that were previously unanticipated. In this work, I and other members of the Gilbert laboratory worked to characterize the replication program in mammalian development, and described its structure, developmental regulation, and potential applications to medicine. In genome-wide studies of replication timing in mice, we found that replication timing profiles are both remarkably stable and cell type-specific, and are composed of coordinately regulated units (replication domains) that span one to several megabases. Changes in replication time typically occurred in 400-800kb units, and encompassed roughly 20% of the genome upon differentiation of embryonic stem cells (ESCs) to neural precursor cells (NPCs). These changes remarkably aligned domain timing values to genomic GC content and LINE-1 retrotransposon density. Consistent with previous results at individual loci, early replication was significantly (but not perfectly) associated with active transcription and active histone marks, and switches to later and earlier replication were accompanied by chromatin movement toward and away from the nuclear periphery respectively. Since H3K9 dimethylation was the only repressive histone mark with a moderate relationship to late replication, we next studied the regulation of the replication program with a cell line harboring an inducible conditional knockout of histone methyltransferase G9a. However, by comparison to the typical amount of timing differences between replicates (roughly 2-4% of the genome), we found no regions exhibiting unusually large timing changes upon G9a knockout in ESCs, or after differentiation of G9aCKO cells into NPCs. Nevertheless, many late-replicating H3K9me2-marked genes were transcriptionally upregulated, providing evidence that partially uncouples expression and histone mark changes from replication timing and nuclear location. To determine the extent of conservation between mouse and human replication program, we profiled several human ESC lines, differentiated NPCs, and lymphoblasts. Nearly all of the major properties from mouse were consistent in human cells, including domains sizes, timing changes in 400-800kb units, and relationships to activating histone marks and transcription. We also demonstrated that the replication program is well-conserved in regions syntenic between human and mouse. Importantly, hESCs aligned most closely not to mESCs, but to mouse EpiSCs, a more advanced population of cells derived from the epiblast and with comparatively limited plasticity. In studying the relation to histone marks we observed a peak of active marks 100kb within the border of most early replicating domains, but most remarkably (and unexpectedly), we found a correlation between replication timing profiles and Hi-C chromatin interactions stronger than any other genomic property, despite the Hi-C data deriving from an abstract computational model. As the robust cell type specificity of replication profiles suggested their potential for use in cell typing and studies of disease, I created (in collaboration with Jinfeng Zhang) a computational method to define "replication fingerprints"--collections of genomic regions with unique patterns of replication in defined collections of samples. Using these regions, 67/67 (34 mouse and 31 human) datasets could be correctly classified among 11 mouse and 9 human tissue types using a simple nearest-neighbor approach, and these results were confirmed through cross-validation and independent PCR assays. As a biological application, we created a fingerprint to isolate regions with common timing changes between pluripotent and committed cells, which revealed a conserved switch to later replication in the major histone H1 cluster that may help to explain the chromatin compaction previously observed during differentiation. To apply what we have learned about the replication program to the study of human disease, we collaborated with Drs. Bill Chang and Brian Druker to profile cell lines and pediatric patients with acute lymphoblastic leukemia (ALL). In contrast with normal B and T lymphocytes, leukemic samples displayed a high level of heterogeneity in replication profiles that offered intriguing potential for epigenetic fingerprints. Therefore, we applied the fingerprinting method to define regions with unique replication timing in high-risk patients and various genetic subtypes. To confirm the identity of leukemic samples and ability to detect known genetic lesions, we identified translocations and copy number variants in cell lines and patient samples known from CGH or karyotype information. These studies have opened paths to study less well-characterized subtypes of leukemia such as AML, which we plan to explore in future work. Show less

Date Issued

2012

Identifier

FSU_migr_etd-5153

Format

Thesis

Title

Cell Signaling and the Regulation of Axis Formation, Cell Proliferation, and Differentiation in Drosophila Melanogaster.

The development of multicellular animals involves a diverse array of cellular processes, including cell differentiation, proliferation, and polarization. The control of these processes is largely governed by communication between different cells. This intercellular communication, known as cell signaling, is therefore a fundamental aspect of developmental and cellular biology. Despite a wealth of knowledge regarding the canonical cell signaling pathways, many questions remain regarding the... Show moreThe development of multicellular animals involves a diverse array of cellular processes, including cell differentiation, proliferation, and polarization. The control of these processes is largely governed by communication between different cells. This intercellular communication, known as cell signaling, is therefore a fundamental aspect of developmental and cellular biology. Despite a wealth of knowledge regarding the canonical cell signaling pathways, many questions remain regarding the mechanistic nature of the communication taking place during specific developmental events, as well as questions regarding the control of activation of cell signaling. In this dissertation I will use the egg chamber of Drosophila melanogaster as a model system to investigate the genetics and cellular biology surrounding two important developmental events involving cell signaling. In the first part I describe a role for an adhesion molecule, Dystroglycan (DG), in the communication between two important cell types present in the egg chamber (the follicle cells and the oocyte). This communication is of great developmental significance because it creates the foundation for the polarization of the oocyte. The finding that DG is involved in this process suggests that changes in cell adhesion are important in the communication that establishes oocyte polarity. In the second part of the dissertation I identify a novel role for the gene, Belle (Bel), in controlling the activation of a key cell signaling pathway known as Notch. Notch activation in the follicle cells is essential for many aspects of egg chamber development. I also demonstrate that the regulation of Notch by Bel occurs through Bel's role in the microRNA pathway, possibly through regulation of levels of another protein, Delta. Together my research sheds new light on two key facets of egg chamber development that will potentially elucidate similar mechanisms present in other aspects of development in Drosophila, as well as other organisms. Show less

This research focuses on the characterization of three isoforms of the human cardiac Troponin C protein previously designed and created by Myriam Badr. These isoforms differ both in the fluorophores bound to the N and C termini and the size of the linker sequences between the fluorophores and TnC protein. Troponin C is notable for its function in Ca2+ binding, which, in functioning muscle, induces a conformational change. This reveals the hidden myosin head binding site and allows contraction... Show moreThis research focuses on the characterization of three isoforms of the human cardiac Troponin C protein previously designed and created by Myriam Badr. These isoforms differ both in the fluorophores bound to the N and C termini and the size of the linker sequences between the fluorophores and TnC protein. Troponin C is notable for its function in Ca2+ binding, which, in functioning muscle, induces a conformational change. This reveals the hidden myosin head binding site and allows contraction to proceed. Using fluorometer analysis to measure changes in FRET, the structural changes each undergoes in the presence of calcium, magnesium, and calcium in the presence of magnesium were analyzed to better understand the functioning of each of these proteins under physiological conditions. Since deficits in cardiac troponin C function have been implicated in some cases of familial hypertrophic cardiomyopathy, it is important to better understand how function changes with the size and shape of the cardiac troponin C protein. With this in mind, we hope to gain a more complete understanding of human cardiac troponin C function. Show less

Date Issued

2016

Identifier

FSU_2016SP_Lentsch_fsu_0071N_13055

Format

Thesis

Title

Characterization of Dopamine and Kainate Receptors in Olfactory Bulb Neurons and Their Efffects on Glutamatergic Transmission.

The main olfactory bulb (OB) receives odorant information from the nasal epithelium, interprets much of that information, and transmits the results to higher cortical regions. The predominant excitatory neurotransmitter in the OB and throughout the brain is glutamate. Modulators of glutamatergic activity influence synaptic transmission of intrabulbar circuits profoundly; therefore, the effects of neuromodulators must be thoroughly characterized in order to understand fully how OB circuits... Show moreThe main olfactory bulb (OB) receives odorant information from the nasal epithelium, interprets much of that information, and transmits the results to higher cortical regions. The predominant excitatory neurotransmitter in the OB and throughout the brain is glutamate. Modulators of glutamatergic activity influence synaptic transmission of intrabulbar circuits profoundly; therefore, the effects of neuromodulators must be thoroughly characterized in order to understand fully how OB circuits function. Investigations performed here address the capacity of dopamine receptor (DAR) and kainate receptor (KAR) activation to modulate glutamate transmission from principal cells to interneurons in OB primary cultures. Initially, I obtained immunocytochemical evidence for DARs expressed in principal cells. Subsequent electrophysiological analyses revealed that the D2-like receptor subtype (D2Rs) attenuated both spontaneous and evoked glutamatergic transmission. Information gleaned from studies of input resistances and calcium currents allowed me to determine that the site of modulation is located on the presynaptic cell. My research into KARs demonstrated the existence of functional KARs in OB neurons and began to elucidate their physiological roles in OB neurotransmission. First, I gathered immunocytochemical evidence to visualize KARs expressed both at and near synapses. In situ hybridization (ISH) was employed to map which OB neurons express mRNA for each KAR subunit. Expression levels for each subunit were quantified in parallel studies using the reverse transcriptase-polymerase chain reaction (RT-PCR). Electrophysiological approaches were used to determine whether or not KARs participate in synaptic transmission between OB neurons in primary cultures. I provide evidence for KAR-mediated modulation of both spontaneous and evoked glutamatergic transmission between OB neurons. Taken together, this work supports the notion that synaptic transmission of OB neurons can be modulated by either metabotropic or ionotropic ligand-gated ion channels. In addition, this is the first thorough characterization of KAR expression and physiology in OB neurons. Show less

Drosophila melanogasterfemale fruit flies possess a pair of ovaries in which many egg chambers are produced, each containing an oocyte that houses the haploid female gamete. In the process of oogenesis, the multi-cellular egg chamber is composed of somatic follicle cells which encapsulate the germ-cells (1 oocyte and 15 nurse cells); the germ-line nurse cells produce an enormous amount of RNAs and proteins that are needed for growth and morphological changes of the egg chamber to aid in the... Show moreDrosophila melanogasterfemale fruit flies possess a pair of ovaries in which many egg chambers are produced, each containing an oocyte that houses the haploid female gamete. In the process of oogenesis, the multi-cellular egg chamber is composed of somatic follicle cells which encapsulate the germ-cells (1 oocyte and 15 nurse cells); the germ-line nurse cells produce an enormous amount of RNAs and proteins that are needed for growth and morphological changes of the egg chamber to aid in the development of the oocyte in preparation for fertilization and embryogenesis. Intriguingly, nurse-cell (NC) nuclei undergo the endocycle in which DNA is re-replicated in the absence of mitosis, creating visible chromatin structures. During stages 4-6 of oogenesis, the NC nuclei undergo a dramatic change in morphology from a visible polytenic state to a diffuse state via a transient condensation phase. Mutations in many genes involved in various transcriptional, splicing, and translational processes routinely retain nurse-cell chromatin dispersal (NCCD) failure phenotypes, in which NC chromatin never disperse in the later stages of oogenesis. However, the significance of NCCD remains elusive in terms of its effect on essential processes like ribosomal synthesis of proteins or oocyte polarization. In order to investigate in greater detail the conditions required for NCCD, I first identify the novel genepolythrough a traditional FLP-FRT mosaic clone screen and characterize its requirement in the germ-line for NCCD and oocyte polarization. Later on, I also demonstrate that that NCCD itself does not affect oocyte polarity, through studies of anovarian tumormutation haploinsufficient for NCCD. However, performing a genetic modifier screen with theovarian tumormutation yields many loci that affect the morphology of NC chromatin, suggesting that NCCD is sensitive to genetic background. In the third part, I characterize mutations inpeanuts, a spliceosomal protein isolated from the screen, and its role in mediating spliceosomal function and NCCD, thus validating the screen as a tool for further identification of genes that affect chromatin dynamics. Show less

Macro histone H2A (macroH2A) is a variant of core histone H2A that differs primarily by an extensive carboxy-terminal tail of unknown function that makes up two-thirds of the protein's mass. The histone variant is distributed throughout the nucleus, but in female mammalian cells, it has been found to be associated with the inactive X chromosome (Xi) in a local accumulation referred to as a macrochromatin body. The association of macroH2A with facultative heterochromatin of the Xi is... Show moreMacro histone H2A (macroH2A) is a variant of core histone H2A that differs primarily by an extensive carboxy-terminal tail of unknown function that makes up two-thirds of the protein's mass. The histone variant is distributed throughout the nucleus, but in female mammalian cells, it has been found to be associated with the inactive X chromosome (Xi) in a local accumulation referred to as a macrochromatin body. The association of macroH2A with facultative heterochromatin of the Xi is suggestive of a role for the variant in gene silencing. MacroH2A1 was the first form of macroH2A discovered and is encoded by the H2AFY gene. Two splice isoforms exist due to two alternate versions of exon-6, giving rise to macroH2A1.1 and macroH2A1.2. A second form of macroH2A encoded by H2AFY2 gene, known as macroH2A2, shares 80% amino acid identity with macroH2A1 and also accumulates at Xi, suggesting the possibility of functional redundancy between the two proteins. In order to further investigate macroH2A, we have generated knockouts of macroH2A1 and targeted a single allele of macroH2A2 in a human female telomerase immortalized retinal pigment epithelial cell line (RPE1). Targeted clones were generated by exchanging exon-2 of one or both alleles with a promoter-trap construct containing a promoterless neomycin selection cassette flanked by arms of homology designed to the intronic sequences immediately adjacent to exon-2. The selection cassette contains a splice-acceptor, internal ribosome entry site and polyadenylation signal, which when exchanged with exon-2 results in the inclusion of the neomycin coding sequence in the resulting truncated messenger RNA and its subsequent translation, allowing for correct targeting to be selected for through neomycin resistance. To enhance targeting, Zinc Finger Nucleases (ZFNs) were engineered to create a double strand break at or close to exon-2. In order to target the genes, cells were co-nucleofected with the targeting construct and ZFNs before seeding cells in media containing neomycin. Single cell clones were screened for correct targeting and loss of macroH2A assessed by Western blotting. In order to explore the impact of macroH2A1 loss, RNA was extracted from a macroH2A1 knockout as well as parental RPE1 and changes to the transcriptome assessed by massively paralleled sequencing of complementary DNA (RNAseq). Genes that showed a significant change in expression between the wildtype and knockout cells were selected for further study. Quantitative Chromatin Immunoprecipitation (qChIP) was performed on an affected gene to evaluate any local chromatin changes due to macroH2A1 loss. Additionally, to examine the possibility that macroH2A1 splice isoforms fulfill different roles, full-length MYC-tagged expression constructs for macroH2A1.1 and macroH2A1.2 were reintroduced into cells and rescue of wild-type expression assessed for genes that displayed altered expression in response to macroH2A1 loss. Show less

Date Issued

2014

Identifier

FSU_migr_etd-8853

Format

Thesis

Title

Characterization of Role of LARP6 Phosphorylation in Regulating Type I Collagen Biosynthesis in Liver Fibrosis.

Liver fibrosis is the common end stage of all chronic liver diseases, such as chronic viral hepatitis, alcoholism, nonalcoholic fatty liver disease, autoimmune hepatitis, alpha 1 anti-trypsin deficiency and some rare metabolic diseases. Fibrosis it is a major cause of morbidity and mortality worldwide. However, the specific and efficient anti-fibrotic therapy is still lacking. Thus, better understanding the underlying mechanism of liver fibrosis is critical in order to find a cure. Liver... Show moreLiver fibrosis is the common end stage of all chronic liver diseases, such as chronic viral hepatitis, alcoholism, nonalcoholic fatty liver disease, autoimmune hepatitis, alpha 1 anti-trypsin deficiency and some rare metabolic diseases. Fibrosis it is a major cause of morbidity and mortality worldwide. However, the specific and efficient anti-fibrotic therapy is still lacking. Thus, better understanding the underlying mechanism of liver fibrosis is critical in order to find a cure. Liver fibrosis is histologically characterized by excessive deposition of extracellular matrix composed primarily of type I collagen. Type I collagen is a complex protein composed by folding by two α1(I) and one α2(I) polypeptides into triple helix. The production of collagen polypeptides is regulated by the cis-acting sequence of their respective mRNAs, the 5' stem loop (5'SL). In the 5' untranslated region of collagen α1(I) and α2(I) mRNAs, there is a secondary structure forming a stem loop (5'SL). This cis-acting element regulates type I collagen expression in fibrosis by binding an RNA binding protein, La ribonucleoprotein domain family, member 6 (LARP6). LARP6 specifically binds to 5'SL of collagen mRNAs with high affinity and sequence specificity. The binding recruits several effector proteins to stimulate type I collagen production in fibrosis. LARP6 is a phosphor-protein, however, how the phosphorylation of LARP6 is involved in the process of collagen biosynthesis has not been studied before. My dissertation focuses on the role of LARP6 phosphorylation in biosynthesis of type I collagen in fibrosis. I have identified eight serines of LARP6 that undergo phosphorylation and six of these serines have never been reported to be phosphorylated before. I have characterized the functional consequence of phosphorylation of these serines, identified the responsible kinases, and analyzed the role in collagen biosynthesis. These studies are presented in the dissertation as three logically connected chapters. In the chapter two provide evidence that phosphorylation of LARP6 follows a hierarchical order; namely, that phosphorylation of S451 is the initial event, which is required for phosphorylations of other serines. Phosphorylation of S451depends on the activity of PI3K/Akt signaling pathway. Akt inhibitor, GSK-2141795, which is in clinical trials for treatment of solid tumors, reduced collagen production with EC50 of 150 nM. This effect is explained by inhibition of LARP6 phosphorylation and suggests that Akt inhibitors may be effective in treatment of xi various forms of fibrosis. The S451A mutant of LARP6 lacks phosphorylation, not only at 451 position, but also at several other serines. Its overexpression has a dominant negative effect on collagen biosynthesis; the S451A mutant drastically reduces secretion of type I collagen and induces synthesis of aberrant and over-modified collagen polypeptides. This indicates that LARP6 phosphorylation at S451 is critical for activation of the protein in translation and folding of collagen polypeptides. In the chapter three I have characterized two other phosphorylations of LARP6, the phosphorylation of S348 and S409. These sites are phosphorylated by mTORC1 and are redundant. Mutation of both of these serines is required to inactivate LARP6. The double mutant, S348A/S409A, acts as a dominant negative protein in collagen biosynthesis, which retards secretion of type I collagen and causes excessive posttranslational modifications. Similar effects are seen using mTORC1 inhibitor rapamycin or by knocking down mTORC1 function by siRNA. The phosphorylation of S348A or S409A is needed for two processes: 1. To recruit an accessory protein STRAP to collagen mRNAs and 2. To enable normal subcellular trafficking of LARP6. STRAP is needed to coordinate translation of collagen α1(I) and α2(I) mRNAs, what becomes critical in fibrosis. In the absence of S348/S409 phosphorylation LARP6 is also sequestered in increasing amounts at the ER membrane. The mechanistic details and significance of the S348/S409 phosphorylation are described in chapter 2. The role of TGF-β1 in LARP6 phosphorylation is described in the fourth chapter. TGF-β is the most potent profibrotic cytokine and this discovery provides the link between the TGF-β activity and the LARP6 dependent mechanism of collagen synthesis. The phosphorylation of LARP6 at S396 is stimulated by TGF-β and it promotes the distribution of LARP6 into the nucleus. This is necessary for binding of the newly transcribed collagen mRNAs and their inclusion in the LARP6 dependent metabolic pathway, resulting in more efficient type I collagen expression. In conclusion, my dissertation work has characterized different phosphorylation events of LARP6 and how they are involved in regulating the function of LARP6 in type I collagen biosynthesis. These findings will contribute to better understanding of the underlying mechanism of overproduction of type I collagen in fibrosis, and provide the rationale of using kinase inhibitors for treating fibrotic disorders. Show less

Date Issued

2016

Identifier

FSU_2016SP_Zhang_fsu_0071E_13114

Format

Thesis

Title

Characterization of the Cytosolic Proteins Involved in the Amoeboid Motility of Ascaris Sperm.

The amoeboid sperm of Ascaris crawl through a cycle of protrusion, adhesion, and retraction, similar to that seen in conventional actin-based cells. However, instead of actin, these cells power their movement through modulation of the major sperm protein (MSP) cytoskeleton. MSP forms dense filament meshworks that pack the sperm lamellipod. Protrusion is associated with the assembly of MSP filaments at the leading edge of the lamellipod, and retraction is connected with the disassembly of the... Show moreThe amoeboid sperm of Ascaris crawl through a cycle of protrusion, adhesion, and retraction, similar to that seen in conventional actin-based cells. However, instead of actin, these cells power their movement through modulation of the major sperm protein (MSP) cytoskeleton. MSP forms dense filament meshworks that pack the sperm lamellipod. Protrusion is associated with the assembly of MSP filaments at the leading edge of the lamellipod, and retraction is connected with the disassembly of the MSP network at the base of the lamellipod. The motility of Ascaris sperm can be reconstituted in cell-free extracts. In vitro, plasma membrane vesicles are pushed forward by the elongation of fibers constructed from a columnar meshwork of MSP filaments. This in vitro motility requires components from both the cytosol and the vesicle. LeClaire et al. (2003) recently identified the 48 kDa membrane protein required to orchestrate MSP cytoskeletal assembly at the leading edge of the lamellipod. In this study, I describe the first cytosolic proteins that are components of the MSP locomotory machinery. I fractionated cytosol with a range of biochemical techniques and reconstituted fiber assembly with a limited subset of cytosolic components. Thus, this fraction contains all the cytosolic accessory proteins required to build fibers. Several of the components in this active fraction were used to generate antibodies, which labeled the cytoskeleton in Ascaris sperm and in fibers grown in vitro and thus, identified six proteins, p43, p42, p40, p38, p34, and p16, as part of the MSP cytoskeleton. Sequence analysis showed that each protein is novel to nematode sperm and has a homolog of unknown function in C. elegans that exhibits sperm-enriched expression (Reinke et al., 2000; Hill et al., 2001). The predicted protein sequence of p43 is based on a tandem array of repeats with serine phosphorylation sites. P38, p40, and p42 appear to be a family of closely related polypeptides. The sequences of p38 and p40 contain a domain duplication as well as proline-rich repeats. P34 shows homology to serine/threonine kinases. The p16 triplet is a family of closely related polypeptides. Two of the proteins had reciprocal effects on fiber growth: p38 increased fiber growth rate and p16 decreased fiber growth rate. The effects of both p38 and p16 were concentration-dependent and antagonistic. Since the rate-enhancement by p38 was not potentiated by MSP, another cytosolic protein is involved in up-regulation of the rate of MSP elongation. Additionally, p16 altered the number of filaments polymerized at the vesicle surface and thus may regulate MSP nucleation. Immunoprecipitations and affinity chromatography established that these two proteins bind to MSP under conditions that promote in vitro assembly. Based on these data, I present a revised model for the mechanism of membrane-associated MSP polymerization, which proposes that p38 influences the rate of MSP elongation by recruiting MSP to the vesicle surface and that p16 incorporates into MSP filaments and blocks further assembly by acting as a capping protein. My results imply that although the specific components differ, actin-based and MSP-based systems both rely on the interplay of positive and negative regulators of cytoskeletal assembly to maintain the pace of locomotion. Show less

Date Issued

2003

Identifier

FSU_migr_etd-2403

Format

Thesis

Title

Characterization of the Interaction Between Titn Kinase Domain and Enigma/Pdlim7.

Titin is a very large protein that contributes to sarcomere structure and mechanosensing in striated muscle. Our lab discovered isoforms of titin in nonmuscle cells (Eilersten and Keller, 1992). Nonmuscle cell titin (c-titin) contains an alpha-actinin binding Z-repeat region, a distinctly PEVK region, a myosin filament-binding region, and the kinase domain also present in striated muscle titin isoforms. In striated muscle, the titin kinase domain (TKD) functions as a mechanosensor that... Show moreTitin is a very large protein that contributes to sarcomere structure and mechanosensing in striated muscle. Our lab discovered isoforms of titin in nonmuscle cells (Eilersten and Keller, 1992). Nonmuscle cell titin (c-titin) contains an alpha-actinin binding Z-repeat region, a distinctly PEVK region, a myosin filament-binding region, and the kinase domain also present in striated muscle titin isoforms. In striated muscle, the titin kinase domain (TKD) functions as a mechanosensor that signals changes in gene expression through interaction with nbr1 and p62. A previous yeast two hybrid (Y2H) screen to identify proteins that interact with the TKD in nonmuscle cells revealed an interaction with the ubiquitously expressed scaffold protein Enigma/PDLIM7. Enigma/PDLIM7 consists of an N-terminal PDZ domain that binds to β-tropomyosin on actin filaments, a Mid piece, and C-terminal region containing three LIM domains. The work described here further characterizes the interaction between the TKD and Enigma/PDLIM7. Y2H analysis with cloned TKD and Enigma/PDLIM7 fragments demonstrated that a region of the Enigma/PDLIM7 Mid piece and LIM1 and LIM3 domains interact with TKD. In vitro pull-down assays with bacterially expressed protein confirmed the interaction between TKD and Enigma LIM3. Immunolocalization of the TKD and Enigma/PDLIM7 in cultured human mesenchymal stem cells containing robust stress fibers revealed that both TKD and Enigma localized along stress fibers where they could interact, but there was little direct overlap in the cells under the standard culture conditions tested. These results support the possibility that Enigma/PDLIM7 functions as a scaffold to localize the TKD near actin filaments in the cytoskeleton of nonmuscle cells. Show less

Cilia are recognized as an important sensory structure for the cell. However, much about the signaling involved in ciliary outgrowth remains a mystery. Chlamydomonas, a green algae, is a model for ciliary and flagellar studies owing to its pair of anterior flagella and haploid genome. In a previous study of gene expression during flagellar outgrowth in Chlamydomonas after environmental shock, the gene encoding a predicted seven transmembrane protein (Cr7TM) was differentially expressed.... Show moreCilia are recognized as an important sensory structure for the cell. However, much about the signaling involved in ciliary outgrowth remains a mystery. Chlamydomonas, a green algae, is a model for ciliary and flagellar studies owing to its pair of anterior flagella and haploid genome. In a previous study of gene expression during flagellar outgrowth in Chlamydomonas after environmental shock, the gene encoding a predicted seven transmembrane protein (Cr7TM) was differentially expressed. Subsequent experiments described here have attempted to elucidate the function of Cr7TM in the cell and specifically in the flagella. Bioinformatic search techniques and predictions of the evolutionary history of Cr7TM and its homologues imply that this LUSTR protein family is unique in both its primary structure and mechanisms for protein interaction. It is also highly conserved and likely involved in a critical cellular function. LUSTR proteins are ubiquitously expressed across metazoan organisms and tissue types. Also, Cr7TM is differentially expressed during outgrowth of cilia/flagella but not during oxidative stress, indicating Cr7TM's involvement in flagellar outgrowth rather than the stress response. Fluorescence microscopy indicates that Cr7TM co-localizes with acetylated á-tubulin in the flagella. Creation of an inducible knockdown mutant line of Cr7TM allowed for comparison of the phenotypes between induced and uninduced RNAi knockdown mutant lines. The Cr7TM knockdown line demonstrates a diminished cell size along with a rapid increase in the density of cell cultures as compared to the control, indicating a possible cellular role for Cr7TM in cell cycling or mitotic regulation. Additionally, imaging of human embryonic kidney cells shows colocalization of LUSTR proteins near the highly conserved centriole and basal body organelles. When taken together the high degree of sequence conservation, knockdown phenotype and cellular localization of LUSTR proteins provide evidence for LUSTR involvement in the regulation of cell cycle. Show less

Date Issued

2012

Identifier

FSU_migr_etd-5800

Format

Thesis

Title

The Characterization of the Roles of Ipl1 and Cdc5 in Spindle Pole Body Duplication in Yeast Meiosis.

The centrosome is the microtubule organizing center (MTOC) in higher eukaryotes. During meiosis, proper duplication and separation of the centrosomes are necessary for accurate chromosome segregation and leads to the production of gametes containing half of the parental genome. During meiotic interphase I, centrosomes are duplicated when chromosomes replicate. A pair of centrosomes establish a bipolar microtubule spindle that facilitates the segregation of homologs during meiosis I.... Show moreThe centrosome is the microtubule organizing center (MTOC) in higher eukaryotes. During meiosis, proper duplication and separation of the centrosomes are necessary for accurate chromosome segregation and leads to the production of gametes containing half of the parental genome. During meiotic interphase I, centrosomes are duplicated when chromosomes replicate. A pair of centrosomes establish a bipolar microtubule spindle that facilitates the segregation of homologs during meiosis I. Centrosomes duplicate once more at interphase II, when DNA duplication is absent, and form two independent spindles for sister chromatid separation during meiosis II. The centrosome in yeast is called the spindle pole body (SPB). Here we show that the Aurora Kinase Ipl1, which protects sister chromatid cohesion, is also required for the maintenance of a tight association between duplicated sister SPBs, referred to here as SPB cohesion. Premature loss of cohesion leads to SPB over-duplication and the formation of multipolar spindles during meiosis II. The Polo-like kinase Cdc5 is a licensing factor for SPB duplication at interphase II and promotes SPB separation during meiosis II. Our data suggests Ipl1 and Cdc5 interact antagonistically at the SPB to maintain proper duplication and separation of SPBs during meiosis. Show less

Date Issued

2011

Identifier

FSU_migr_etd-1746

Format

Thesis

Title

Characterizing the Relationship Between Williams Syndrome Transcription Factor and Heterochromatin Maintenance Through the Targeted Disruption of the BAZ1B Gene.

Maintaining genome integrity and epigenetic programming is essential to avoid disease and retain the identity and proper function of the multitude of specialized differentiated cell types in the body. Much progress has been made in the past 10 years toward identifying and understanding the range of proteins and complexes involved in these processes. Many of these are chromatin-remodeling complexes, including those that contain the Williams syndrome transcription factor (WSTF). This protein is... Show moreMaintaining genome integrity and epigenetic programming is essential to avoid disease and retain the identity and proper function of the multitude of specialized differentiated cell types in the body. Much progress has been made in the past 10 years toward identifying and understanding the range of proteins and complexes involved in these processes. Many of these are chromatin-remodeling complexes, including those that contain the Williams syndrome transcription factor (WSTF). This protein is particularly interesting for two reasons. First, it functions in many central nuclear processes, such as DNA replication, transcription, and DNA repair. Second, WSTF is haploinsufficient in Williams-Beuren syndrome (WBS) patients. This is because the WSTF gene, BAZ1B, is deleted, along with approximately 27 other genes, from one copy of chromosome 7 in affected individuals. Prior research has implicated WSTF in contributing to several of the phenotypes exhibited in WBS, yet many aspects concerning the function of WSTF remain unclear. A more detailed understanding of WSTF function is necessary to appreciate how this versatile protein contributes to general nuclear processes, and how perturbation of these roles contribute to the symptoms displayed in WBS patients. This study examines the function of WSTF in several ways. First, this research identifies and characterizes the relationship between WSTF and heterochromatin, with a particular focus on facultative heterochromatin of the human inactive X chromosome (Xi). Next, it describes the generation of human cell lines that either lack or are happloinsufficient for WSTF through the generation of heterozygous and homozygous mutant BAZ1B alleles. Using these invaluable model cell lines, this research explores the impact of WSTF reduction or loss on several processes, including heterochromatin maintenance, the DNA damage response, and vitamin D induced gene expression. This work reveals that WSTF is necessary to maintain appropriate expression of a substantial number of genes, and describes a novel nuclear phenotype in BAZ1B knockout cells, characterized by the spontaneous formation and subsequent resolution of extensive regions of heterochromatin throughout the nucleus. This research contributes to and extends current understanding of WSTF function, and provides BAZ1B knockout cells to further investigate WSTF mechanism, as well as providing BAZ1B heterozygous knockouts that will serve as a model to examine how WSTF haploinsufficiency contribute to WBS in the absence of the effects of the other 27 genes that are typically deleted in the disorder. Analyses found in this dissertation link WSTF function to maintenance of chromatin and transcriptional states. Through the examination of BAZ1B knockout cells, this work also underscores that the current understanding of WSTF is not as clear as anticipated, given that processes expected to be disrupted in the absence of WSTF were unaffected. This dissertation concludes with a discussion of these findings as well as future implications. Show less

Rodents depend on the main olfactory and accessory olfactory systems to detect and process chemical-communication signals important for complex reproductive and social behaviors. As the medial amygdala is the first site of convergence of these two pathways in the brain, examining its role in chemosensory processing is of primary importance. Previous experiments have shown that the hamster medial amygdala exhibits a categorical response to different types of chemical signals. The use of... Show moreRodents depend on the main olfactory and accessory olfactory systems to detect and process chemical-communication signals important for complex reproductive and social behaviors. As the medial amygdala is the first site of convergence of these two pathways in the brain, examining its role in chemosensory processing is of primary importance. Previous experiments have shown that the hamster medial amygdala exhibits a categorical response to different types of chemical signals. The use of immediate early gene (IEG) expression to assess neural activity in different brain regions showed that chemical signals from the same species (conspecific) activated anterior and posterior medial amygdala, while chemical signals from other species (heterospecific) increased activity in only the anterior medial amygdala. The experiments discussed below aim to provide information about how the mouse medial amygdala responds to categories of chemical signals, how important the main and accessory olfactory systems are to the medial amygdala response and whether oxytocin (which has been shown to be important in a variety of social behaviors) is necessary for the medial amygdala categorization of chemical signals. Upon exposure of male mice to chemical-communication signals, I found that conspecific chemosignals (male, female mouse urine) increased immediate early gene-protein (IEG) expression in both anterior and posterior medial amygdala of male mice, whereas most heterospecific chemosignals (e.g.: hamster vaginal fluid, steer urine) increased expression only in anterior medial amygdala. This categorization of responses in medial amygdala conforms to the previously reported findings in male hamsters. The same characteristic pattern of IEG expression appeared in the medial amygdala of each species in response to conspecific stimuli for that species. These results suggested that the amygdala categorizes stimuli according to the biological relevance for the tested species. Thus, a heterospecific predator (cat collar) stimulus, which elicited behavioral avoidance in mice, increased IEG expression in mouse posterior medial amygdala (like conspecific stimuli). Further analysis suggests reproduction related and potentially threatening stimuli produce increased IEG expression in different sub-regions of posterior medial amygdala (dorsal and ventral, respectively). These patterns of IEG expression in medial amygdala may provide glimpses of a higher-level processing of chemosensory signals beyond the primary-level selectivity of chemosensory neurons, the secondary sorting in main and accessory olfactory bulbs and the tertiary sorting by the medial amygdala into "biologically relevant and non-relevant" categories. Both non-volatile and volatile chemical-communication signals may be detected by the vomeronasal organ, which sends projections to the accessory olfactory bulb and on to the medial amygdala. Results of the first experiment (above) argue that the mouse medial amygdala sorts complex chemosensory information categorically, according to its biological relevance (salience). In order to determine the role of AOS in categorization, male mice underwent vomeronasal removal surgery (VNX) or a sham-operation (SHAM) and then were exposed to conspecific (male and female mouse urine) or heterospecific (hamster vaginal fluid and worn cat collar) chemical stimuli. As with the mice in the above experiment, SHAM mice exhibited different IEG expression patterns in the medial amygdala dependent upon the biological relevance of the chemical stimuli. However, regardless of biological relevance, vomeronasal organ removal eliminated the different IEG response patterns in the medial amygdala to any of the chemical stimuli. Interestingly, VNX also disrupted the avoidance of (an unfamiliar) predator odor, worn cat collar. These experiments show that the medial amygdala response to these tested chemical signals is dependent upon an intact vomeronasal organ. Normal function of the neuropeptide oxytocin (OT) in the medial amygdala is necessary for social recognition. In the final set of experiments, male mice having undergone intracerebroventricular cannulation (i.c.v.) were injected with either PBS (control) or oxytocin antagonist (OTA) and exposed to conspecific (female mouse urine) and heterospecific (steer urine and worn cat collar) chemical stimuli. As in the above experiments, PBS-injected mice exhibited different IEG expression patterns in the medial amygdala dependent upon the biological relevance of the chemical stimuli. However, OTA injection eliminated the increase in IEG expression in the medial amygdala to all of the tested conspecific or heterospecific stimuli. Importantly, OTA injection disrupted defensive and non-defensive behaviors after exposure to the unfamiliar predator odor, worn cat collar. The disruption of the social/individual recognition behavior in male mice deficient in OT may be due to the inability of the medial amygdala to process all biologically relevant chemical-communication signals. Show less

Nucleosome distributions are critically important in regulating access to the eukaryotic genome. Cells with different physiologies have strikingly similar nucleosome distributions. Few studies in human cells have measured genome-wide nucleosome distributions at high temporal resolution during a response to a common stimulus. Factors regulating the maintenance of the basal state as well as changes in nucleosome distribution following a response must be investigated. In our first set of... Show moreNucleosome distributions are critically important in regulating access to the eukaryotic genome. Cells with different physiologies have strikingly similar nucleosome distributions. Few studies in human cells have measured genome-wide nucleosome distributions at high temporal resolution during a response to a common stimulus. Factors regulating the maintenance of the basal state as well as changes in nucleosome distribution following a response must be investigated. In our first set of experiments we used the reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) as a model system for stimulus-induced nucleosome distribution changes. We measured nucleosome distribution at high temporal resolution in human cells at the 2 kb flanking the transcription start sites (TSSs) of hundreds immunity-related loci, using microarray technology, during the reactivation of KSHV. We show that nucleosome redistribution peaks at 24 hours post KSHV reactivation and that the nucleosomal redistributions are widespread and transient. To clarify the role of DNA sequence in these nucleosomal redistributions, we compared the genes with altered nucleosome distribution to a sequence-based computer model and in vitro assembled nucleosomes. We demonstrate that both the predicted model and the assembled nucleosome distributions are concordant with the majority of nucleosome redistributions at 24 hours post KSHV reactivation. We suggest a model in which loci are held in an unfavorable chromatin architecture and "spring" to a transient intermediate state directed by DNA sequence information. We propose that DNA sequence plays a more considerable role in the regulation of nucleosome positions than was previously appreciated. The surprising findings that nucleosome redistributions are widespread, transient, and DNA-directed shift the current perspective regarding regulation of nucleosome distribution in humans. We next wanted to affirm and extend our previous observations regarding the widespread and transient nature of nucleosome redistributions during viral reactivation. We tested if this widespread nucleosome remodeling was a genome wide event or limited solely to the hundreds of immunity-related loci measured by microarray. We measured nucleosome distributions at high temporal resolution following KSHV reactivation using our newly developed mTSS-seq technology, which maps nucleosome distribution at the TSS of all human genes. Nucleosomes underwent widespread changes in organization 24 hours after KSHV reactivation and returned to their basal nucleosomal architecture 48 hours after KSHV reactivation. 72% of the loci with translationally remodeled nucleosomes have nucleosomes that moved to positions encoded by the sophisticated underlying DNA sequence. We demonstrated that these widespread alterations in nucleosomal architecture potentiated regulatory factor binding. These descriptions of nucleosomal architecture changes have allowed us to propose a new hierarchical model for chromatin-based regulation of genome response. Given that we discovered that nucleosome distributions are widespread and transient, it was important for us to understand the forces maintaining the basal state. We It would be interesting to understand the forces and factors that maintain nucleosome architecture in a basal state and regenerate it following a response, such as KSHV reactivation. An appealing group of candidates that might maintain and regenerate the nucleosome architecture in its basal state is the transcriptional machinery. We identified RNA polymerase II (RNA Pol II) as a likely candidate as it is found throughout the genome and not always associated with transcription. We next were interested in the role RNA Pol II plays in the maintenance of chromatin structure. We measured nucleosome distributions in response to RNA Pol II inhibition by δ-amanitin treatment. Nucleosome distribution changes, following RNA Pol II inhibition, were widespread and the TSSs with nucleosome distribution changes were enriched for RNA Pol II independent of it's role it plays in active transcription. This work gives new insight into understanding the role of chromatin structure regulates and maintains the human genome. Show less

The pituitary gland hormone prolactin (PRL) regulates diverse physiological functions in the female mammal. PRL is secreted into peripheral circulation by lactotrophs in the anterior lobe of the pituitary gland. The primary physiological regulator of PRL secretion is Dopamine (DA). Three populations of neuroendocrine DAergic neurons (NDNs) with cell bodies in the periventricular (PEVN) and arcuate (ARN) nuclei of the hypothalamus release DA. During the 4-5 day estrous cycle of the rat, PRL... Show moreThe pituitary gland hormone prolactin (PRL) regulates diverse physiological functions in the female mammal. PRL is secreted into peripheral circulation by lactotrophs in the anterior lobe of the pituitary gland. The primary physiological regulator of PRL secretion is Dopamine (DA). Three populations of neuroendocrine DAergic neurons (NDNs) with cell bodies in the periventricular (PEVN) and arcuate (ARN) nuclei of the hypothalamus release DA. During the 4-5 day estrous cycle of the rat, PRL secretion peaks on the afternoon of proestrus, due to a gradual rise in circulating ovarian steroids. Experiments show that the proestrous afternoon rise in PRL is timed by inputs from the biological clock located in the suprachiasmatic nucleus (SCN). Studies verify that disruption of the connection between SCN and its targets within the hypothalamus disrupt the timing of PRL secretion. Recently, it has been shown that the oscillatory function of the SCN occurs due to autoregulatory negative feedback loops of transcription factor expression within SCN neurons. These transcription factors are referred to as "clock genes". Clock genes drive cell autonomous oscillations of gene expression and activity in the SCN and additional areas of the CNS, coordinating rhythms of physiological activity. Given that the timing of PRL secretion appears to be regulated by the SCN and that NDNs receive direct input from the SCN, I hypothesized that circadian rhythms of activity in NDNs time PRL secretion in the ovariectomized (OVX) rat. I have shown that NDNs exhibit circadian and semi-circadian rhythms of activity that are modulated by ovarian steroid hormones. Further, I have determined the light-entrained and free-running rhythms of clock gene expression in NDNs. In addition, I have found that antisense knockdown of several clock genes in the SCN modulates, but fails to abolish, circadian rhythms of NDN activity. Results from these experiments, in agreement with previous work, reveal that NDNs display circadian and semi-circadian rhythms of DA release, driven by direct influence from the SCN. My results suggest a functional link between the expression of clock genes within SCN neurons and NDNs in the control of circadian rhythms of DA release and PRL secretion in the female mammal. Show less

Ecological systems are dynamic, yet many experimental studies examine plant-herbivore interactions as from a simple, static, or single perspective. Reciprocal interactions can have profound effects on communities, and ignoring such feedbacks can result in mismatches between theoretical predictions and experimental results. In this dissertation, I examined reciprocal interactions between two plant species, Solanum carolinense and Solidago altissima and their insect herbivores. In chapter 2, I... Show moreEcological systems are dynamic, yet many experimental studies examine plant-herbivore interactions as from a simple, static, or single perspective. Reciprocal interactions can have profound effects on communities, and ignoring such feedbacks can result in mismatches between theoretical predictions and experimental results. In this dissertation, I examined reciprocal interactions between two plant species, Solanum carolinense and Solidago altissima and their insect herbivores. In chapter 2, I examined how insect herbivores influenced plant competition and coexistence. Theory suggests that herbivores influence plant communities by altering competitive interactions. Because the outcome of competition is influenced by both the per capita competitive ability of plants and demographic processes including density dependence and intrinsic population growth rates, measuring herbivore effects on all these processes is necessary to understand the mechanisms by which herbivores influence plant communities. I fit alternative competition models to data from a response surface experiment conducted over four years to examine how herbivores affected the outcome of competition between two perennial plants, Solanum carolinense and Solidago altissima . Within a growing season, herbivores reduced Solanum plant size, but did not affect Solidago, which exhibited compensatory growth. Across seasons, herbivores did not affect the density of Solanum but reduced both the density and population growth of Solidago. The best fit models indicated that the effects of herbivores varied with year. In some years, herbivores increased the per capita competitive effects of Solidago on Solanum; in other years herbivores influenced the intrinsic population growth rates of Solidago. I examined herbivore effects on the longer-term outcome of competition (over the time-scale of a typical old-field habitat) using simulations based on the best fit models. In the absence of herbivores, plant coexistence was observed. In the presence of herbivores, Solanum was excluded by Solidago in 60% of the simulations. I demonstrated that herbivores can influence the outcome of competition through both changes in per capita competitive effects and changes in demographic processes. I discuss the implications of these results for ecological succession and biocontrol. In chapter 3, I examined how plant community composition influenced damage patterns on plants. Neighboring plants can increase (associational susceptibility) or decrease (associational resistance) the likelihood of damage to a focal plant but their long-term consequences for plant competition and coexistence are unclear. Neighbor effects on damage can occur through changes in the relative density of the focal plant (i.e., frequency of the focal plant), the absolute density of the focal plant, or through the total density of plants, because the different mechanisms known to influence damage patterns (e.g., pest suppression by predators, herbivore foraging behavior, plant quality) respond to different features of the neighborhood. To examine the long-term consequences of neighbor effects for plant communities, an understanding of how density and frequency of plants influence damage is needed. Using a response surface experimental design, I examined the effects of plant density and frequency on damage to Solanum carolinense. I found non-linear effects of the frequency of heterospecific neighbors (Solidago altissima) on Solanum damage, and a positive effect of Solanum density on damage. The non-linear pattern suggests that multiple mechanisms may be operating to influence damage. Non-linear patterns may be common in other habitats but might be overlooked because traditional neighborhood studies use a very narrow range of densities in their experiments. I encourage future neighborhood studies to use response surface designs to determine the prevalence of non-linear relationships in nature. In chapter 4, I examined how neighborhood composition (i.e. plant density and frequency) influenced four mechanisms known to influence damage to plants (predator suppression, foraging behavior of herbivores, plant quality, and microclimate) using a response surface experimental design. An associational effect was observed between Solanum damage and the frequency of a heterospecific neighbor (Solidago altissima). Predator abundance and richness, soil moisture, and herbivore foraging strategies were all influenced by the frequency of Solidago, suggesting that these mechanisms may contribute to associational susceptibility in this interaction. Other mechanisms (microclimate and plant quality) were influenced by Solidago and total plant densities, respectively. This study showed that different mechanisms can be influenced by different components of the neighborhood and most likely interacts to influence damage to plants. I discuss the implications of these finding for agriculture and for understanding the long-term consequences of damage for plant communities. In chapter 5, I examined how herbivory, herbivore community composition, plant nutrient content, and herbivore performance varied with latitude. A longstanding theory in biogeography is that species interactions, including herbivory, are stronger in southern latitudes compared to those in the north. Because of this, the latitudinal gradients (LG) hypothesis in damage and plant defenses predicts that plants should be better defended in the tropics because selection for plant defenses is greater. Recent empirical studies suggest that the predictions from this hypothesis may be limited to a narrow range of systems (e.g. salt marshes). In efforts to understand why LG in herbivory and plant defenses are not prevalent as once thought, I examined relationships between herbivore abundance and richness, plant nutrient content, and latitude in old-field systems. I also examined latitudinal gradients in herbivore performance using generalist and specialist herbivores. Some relationships with latitude matched predictions from the LG hypothesis (e.g. plant nutrient content, damage to Solidago altissima), while others had opposite relationships (e.g. herbivore abundance and richness, damage to Solanum carolinense), and some relationships varied with leaf longevity. Herbivore responses varied with diet specialization and the exact relationship with latitude (linear, non-linear, positive, negative, or no relationship) varied with herbivore species. These results suggest that the predictions from the LG hypothesis are too simple; a more thorough investigation of relationships between herbivore abundance, damage, and plant resistance in other wide-ranging systems is needed. Show less

Date Issued

2012

Identifier

FSU_migr_etd-5381

Format

Thesis

Title

A Comparison of Invasive and Noninvasive Commelinaceae in a Phylogentic Context.

Predicting which species will become invasive when introduced to a novel range is one of the main goals of invasion biology. However, many studies of the factors influencing invasion either use the wrong comparison group, fail to control for relatedness, or measure traits in only a single environment. I compared invasive and noninvasive species in the Commelinaceae (dayflower family) across multiple environments in the greenhouse and developed a molecular phylogeny of invasive and noninvasive... Show morePredicting which species will become invasive when introduced to a novel range is one of the main goals of invasion biology. However, many studies of the factors influencing invasion either use the wrong comparison group, fail to control for relatedness, or measure traits in only a single environment. I compared invasive and noninvasive species in the Commelinaceae (dayflower family) across multiple environments in the greenhouse and developed a molecular phylogeny of invasive and noninvasive Commelinaceae for comparative analyses. Invasive Commelinaceae had faster growth rates, higher fecundity, thinner leaves, and greater vegetative reproduction than noninvasive relatives. However, whether some traits, such as thin leaves, were associated with invasiveness is environment-dependent, suggesting that predictive models need to take the trait-environment relationship into account to more accurately predict invasiveness. Further, invasive Commelinaceae exhibited opportunistic responses to high nutrient environments, and noninvasive species did not, suggesting that a lack of opportunism may be useful for predicting noninvasive species. Demographic models parameterized with greenhouse data suggest that fecundity (for self-compatible species), time to first reproduction, and vegetative reproduction may be particularly important for explaining the greater performance of invasive taxa in high nutrient environments. Phylogenetic analyses based on trnL-trnF (a non-coding chloroplast region) and 5S NTS (a non-coding nuclear ribosomal repeat) were mostly consistent with tribal and subtribal relationships in the Commelinaceae, and with a previous rbcL phylogeny at the genus level and relationships within genera were mostly well-resolved and consistent with current taxonomy, with the exception of Callisia, which is not monophyletic in these analyses. Comparative tests incorporating phylogeny suggest that weediness, self-compatibility, annual life history, vegetative reproduction, and possibly seed heteromorphism all increase the probability of becoming invasive. In phylogenetically naive tests, capsule dimorphism, autogamous selfing proportion, propagule pressure, and date of first introduction were also associated with invasiveness, although those relationships were not significant in tests incorporating phylogeny. Overall, many traits, including several novel ones presented here (e.g. seed heteromorphism) were associated with invasiveness in the Commelinaceae. Also, environment and phylogeny both influence trait associations, suggesting that taking these factors into account may improve the ability to predict and prevent invasions. Show less

Regular emergence and re-emergence of viral pathogens emphasizes the importance of understanding viral biogeography and migration. Single-stranded DNA (ssDNA) viruses are among the least understood groups of microbial pathogens, yet the group contains known agricultural pathogens, which infect both livestock and crops (Circoviridae and Geminiviridae), and model organisms (Microviridae). Wastewater treatment plants (WWTPs) receive water from multiple sources, becoming reservoirs for the... Show moreRegular emergence and re-emergence of viral pathogens emphasizes the importance of understanding viral biogeography and migration. Single-stranded DNA (ssDNA) viruses are among the least understood groups of microbial pathogens, yet the group contains known agricultural pathogens, which infect both livestock and crops (Circoviridae and Geminiviridae), and model organisms (Microviridae). Wastewater treatment plants (WWTPs) receive water from multiple sources, becoming reservoirs for the collection of many viral families that infect a large range of hosts. Investigations utilizing high-throughput sequencing have determined that local viral diversity is extremely high but does not scale to produce an exponentially higher global diversity. It follows that similar genotypes can be found great distances apart, although they may not be permanent constituents of any single population. Transient genotypes have been observed in temporal surveys of closed systems, where genotypes migrate between individual populations. This study focused on the geographic and temporal population stability of single-stranded DNA (ssDNA) viruses in open systems. Sampling from WWTPs in three neighboring cities in Northwest Florida, which receive constant inflow and potentially receive the same viruses from the local environment, was conducted across a nine-month time span. A combination of polyethylene glycol (PEG) precipitation and filter concentration was used to isolate whole viral particles from the complex wastewater samples. The ssDNA viruses were isolated from larger viruses using a sucrose gradient for size selection and rolling circle amplification was performed to both bias the sample towards ssDNA and prepare the samples for high-throughput sequencing. Amplified genomes were sequenced using Illumina platforms and de novo assembled. Given the increased potential for migration, we expected the populations would be mostly homogenous with relatively few viruses that are unique to individual WWTPs. Viral genotypes with genetic similarity to Circoviridae, Geminiviridae, and Microviridae were recovered from all three WWTPs, however <25% of recovered genes match genotypes (>80% amino acid identity) recovered from neighboring sample sites. We determined that <10% of the genotypes were present in all three plants and the majority of genotypes were specific to one WWTP. Unexpectedly, the WWTPs that were closest to each other geographically were the least similar, and the plants geographically distant from each other had the most observed genetic overlap. These results highlight the high level of diversity within each population, while the high observed heterogeneity indicates localized genetic success and limited migration opportunities between the WWTPs. Throughout time the communities experienced a large degree of genetic turnover. Only 30% of the genotypes were present in more than one time point, 5% were recovered in three of more samplings and <1% were present in all five time points. This thesis concludes that viral genomes are continually moving through the environment and their presence in any given area may be temporary. Therefore, viruses are a continual selective force on their host species through the sheer volume of genetic potential in an area at any given time. Show less

A hallmark of cancerous transformation is altered chromosome structure. Inappropriate regulation of chromatin structure inhibits normal cell function, and may represent the origin of transformation. A handful of important studies have indicated that chromatin structure in the human genome is dynamic and regulatory, but the role of chromatin structure in cancer progression has not been addressed. In our initial studies, we have analyzed chromatin structure of patients with different grades and... Show moreA hallmark of cancerous transformation is altered chromosome structure. Inappropriate regulation of chromatin structure inhibits normal cell function, and may represent the origin of transformation. A handful of important studies have indicated that chromatin structure in the human genome is dynamic and regulatory, but the role of chromatin structure in cancer progression has not been addressed. In our initial studies, we have analyzed chromatin structure of patients with different grades and stages of adenocarcinoma as compared to matched normal tissue using microarray-based nucleosome distribution and chromosomal sensitivity assays. We report that low-grade lung adenocarcinoma displays widespread nucleosome alterations compared to matched normal tissue at over half of 886 genes studied, and that these changes are consistent between patients. Additionally, we determined many changes in early colorectal adenocarcinoma, and the alterations are consistent between patients and concordant with the lung adenocarcinoma alterations. Genes with nucleosome distribution changes are enriched for the phosphoinositide 3-kinase (PI3K) cascade, a key oncoregulatory pathway. Together these results suggest an early, shared regulatory mechanism of transformation. We have also measured substantial disruptions in chromosomal sensitivity in a high-grade and high-stage tumor, linking aggressive tumors with high-order nuclear architecture alterations. From this initial study, we have developed a model in which early adenocarcinoma is linked to changes in nucleosome distributions, whereas aggressive tumors are linked to high-order chromosomal changes. This work has laid the foundation for comprehensive studies on the role of chromatin structure in cancer progression. As such, we have developed a new approach to generate ultra high-resolution, genome-wide nucleosome distribution maps at the transcription start site using deep sequencing, which we call mTSS-seq. Building on the published microarray-based nucleosome distribution mapping of select loci in adenocarcinoma patients, we have provided the first measurements of genome-wide nucleosome distribution using mTSS-seq. We have confirmed that nucleosome distribution is an early, widespread transformation event in lung and colon adenocarcinoma. These findings guide a shift in our perspective from chromatin structural alterations as terminal states to transient events of clinical importance. These altered nucleosome architectures are consistent between patients indicating that they may serve as important early adenocarcinoma markers. We present evidence that the nucleosome alterations are driven by and may influence cis and trans acting factors - the underlying DNA sequence and transcription factor binding. Our study reveals at unprecedented breadth and depth that DNA-directed nucleosome redistributions are a widespread feature early in the progression of cancer, which has allowed us to propose a hierarchical model for genome regulation through chromatin structure. Show less

Date Issued

2014

Identifier

FSU_migr_etd-8771

Format

Thesis

Title

A Computer Simulation Study of the Accuracy of Alternative Maximum-Likelihood Methods for Estimating Ancestral States for Discrete Characters on Phylogenetic Trees.

Parsimony methods have historically been used to reconstruct ancestral character states on a phylogeny in order to test hypotheses about evolution and adaptation. Maximum likelihood methods have recently been considered, with the advantages of quantifying uncertainty and incorporating different models of evolution. However, there have been no conclusive studies as to how much confidence evolutionary biologists should place in maximum likelihood ancestral state reconstruction methods for... Show moreParsimony methods have historically been used to reconstruct ancestral character states on a phylogeny in order to test hypotheses about evolution and adaptation. Maximum likelihood methods have recently been considered, with the advantages of quantifying uncertainty and incorporating different models of evolution. However, there have been no conclusive studies as to how much confidence evolutionary biologists should place in maximum likelihood ancestral state reconstruction methods for discrete binary characters. This study compares the accuracy of two maximum likelihood methods used to reconstruct discrete ancestral character states. In the global method, the transition rate parameters for the likelihood model are estimated only once by maximizing over all states. In the local method, for each node, the node is fixed in a certain state and the transition rate parameters are estimated conditional on that state assignment. In order to examine the accuracy of these two methods, marginal and joint reconstructions were performed on data simulated on a variety of model trees, including those based on phylogenies of real biological organisms, while considering the effects of the model of evolution and the state frequencies at the root of the tree. A custom software program called LASRDISC was written in order to perform all of the necessary simulations and reconstructions for this study. Results show that use of the global and local methods in either marginal or joint reconstructions produce generally the same accuracy rates on average, with both methods having higher accuracy than the parsimony method. There are certain cases when the local method performs better, including when the reconstruction method is too simple, which indicates that the local method is more robust to model violations than the global method. The local method also has higher accuracy when the rates of character evolution are high. The global method has certain advantages also in that for the marginal reconstructions, it is less biased than the local method, leading the local method to be overconfident in the reconstructed probability of a state at a node. Also, the global method is more likely than the local method to produce the same result for marginal and joint reconstructions on the same tree. The major difference in the methods is the time necessary to estimate the reconstructions. There is a significant cost to be considered in terms of speed for a joint reconstruction using the local method, especially for trees with a large number of taxa. Show less

Conditioned taste aversion (CTA) is a unique form of classical conditioning whereby an animal learns to associate a novel taste stimulus with negative visceral effects. Acquisition of CTA results in reduced intake of future presentations of the conditioned novel taste stimulus. Here I investigated both behavioral and neural characteristics of CTA expression in two experiments: 1) taste specificity and learned safety of short-term CTA expression, and 2) whether the same neurons that are... Show moreConditioned taste aversion (CTA) is a unique form of classical conditioning whereby an animal learns to associate a novel taste stimulus with negative visceral effects. Acquisition of CTA results in reduced intake of future presentations of the conditioned novel taste stimulus. Here I investigated both behavioral and neural characteristics of CTA expression in two experiments: 1) taste specificity and learned safety of short-term CTA expression, and 2) whether the same neurons that are activated during CTA expression against conditioned sucrose (the CS) are re-activated by LiCl injection (the US). In the first experiment, we tested whether short-term CTA expression shares with long-term CTA expression the well-defined features of taste specificity to the conditioned taste and learned safety (reduced association of a taste with a toxic effect by prior non-toxic experience with the conditioned taste). We found that short-term expression of CTA was not taste specific and could be attenuated by prior exposure to the conditioned taste only. The second experiment investigated activation at the neural level. CTA acquisition results in expression of c-Fos in the intermediate zone of the nucleus tractus solitaris (NTS) following presentation of the conditioned taste. These patterns of c-Fos expression are similar to what is seen following administration of LiCl. By administering the conditioned taste 3 hours prior to the administration of LiCl, we were able to double-label for c-Fos vii protein (induced by the taste and persisting after mRNA degradation ) and c-fos mRNA (induced by the toxin prior to protein synthesis) and thus measure dual activation of cells by the taste and the toxin. We found that approximately 40% of cells expressing c-Fos were double-labeled for protein and mRNA. Show less

Date Issued

2003

Identifier

FSU_migr_etd-3729

Format

Thesis

Title

Conformation of Regulatory Domain of Myosin in Skeletal and Smooth Muscle.

Reorientation of the regulatory domain of the myosin head is a feature of all current models of force generation in muscle. We have determined the orientation of the myosin regulatory light chain (RLC) using a spin-label bound rigidly and stereospecifically to the single Cys-154 of a mutant skeletal isoform. Labeled RLC was reconstituted into skeletal muscle fibers. Complex electron paramagnetic resonance spectra obtained in rigor necessitated the development of a novel decomposition... Show moreReorientation of the regulatory domain of the myosin head is a feature of all current models of force generation in muscle. We have determined the orientation of the myosin regulatory light chain (RLC) using a spin-label bound rigidly and stereospecifically to the single Cys-154 of a mutant skeletal isoform. Labeled RLC was reconstituted into skeletal muscle fibers. Complex electron paramagnetic resonance spectra obtained in rigor necessitated the development of a novel decomposition technique. The strength of this method is that no specific model for a complex orientational distribution was presumed. The global analysis of a series of spectra, from fibers tilted with respect to the magnetic field, revealed two populations: one well-ordered (±15º) with the spin-label z axis parallel to actin, and a second population with a large distribution (±60 º). A lack of order in relaxed or nonoverlap fibers demonstrated that regulatory domain ordering was defined by interaction with actin rather than with the thick filament surface. No order was observed in the regulatory domain during isometric contraction, consistent with the substantial reorientation that occurs during force generation. For the first time, spin-label orientation has been interpreted in terms of the orientation of a labeled domain. A Monte Carlo conformational search technique was used to determine the orientation of the spin-label with respect to the protein. This in turn allows determination of the absolute orientation of the regulatory domain with respect to the actin axis. Comparison with electron microscopy reconstructions verified the accuracy of the method; the electron paramagnetic resonance determined that axial orientation was within 10 º of the electron microscopy model. In smooth muscle, force generation is regulated by phosphorylation of RLC, which is thought to change the relative positions of myosin heads. To verify this hypothesis, we have measured the distance between selected cysteine mutants of RLC in 6S myosin monomer by conventional and pulse EPR. In the unphosphorylated smooth muscle myosin (SMM) monomer, the distances were determined to be 43% of all RLC sites with distance of 30 Å for SMM Q15C; 36% of 14.7 Å and 22% of 39 Å for SMM N38C; 38% of 30 Å and 21% of 48 Å for SMM S59C; 43% of 35 Å and 33% of 48 Å for SMM T96C; 37% of 33 Å for SMM C108. These results implied that the two RLCs were parallel to each other, leaving the two N domains in close proximity and two C domains far apart. Phosphorylation increased the head-head distance of about 30% SMM N38C and SMM S59C to beyond the limit of EPR measurement. Surprisingly, the distance of two RLCs T96C was still in the range of 18-60 Å. Not only had phosphorylation increased the head-head distance, the width of distance was observed to increase in SMM S59C and T96C. When phosphorylated the rigid conformer of SMM completely vanished and all SMMs become very flexible. Show less

Unraveling the detailed molecular mechanisms involved in muscle contraction is a prerequisite for understanding the molecular basis of muscular disorders. Since myosin is the motor of the muscle contractile system and troponin plays a key role in muscle regulation, this study is aimed at understanding conformational changes occurring in myosin and troponin during muscle activation and force generation. The primary experimental technique used is site specific spin labeling combined with... Show moreUnraveling the detailed molecular mechanisms involved in muscle contraction is a prerequisite for understanding the molecular basis of muscular disorders. Since myosin is the motor of the muscle contractile system and troponin plays a key role in muscle regulation, this study is aimed at understanding conformational changes occurring in myosin and troponin during muscle activation and force generation. The primary experimental technique used is site specific spin labeling combined with electron paramagnetic resonance (SDSL-EPR). Troponin is composed of three subunits: TnC, TnI and TnT. The structure of the inhibitory region of TnI in the troponin ternary complex was determined by measuring the accessibility of spin labels to a water-soluble spin relaxation agent. Results from SDSL-EPR scanning of residues 129 to 145 of cardiac TnI in the ternary complex (C.I.T) suggested that residues 129-137 region fold into an a-helix and residues 138-145 are unstructured. The ternary structure of TnC was determined by measuring distances between different domains of TnC using double electron electron resonance (DEER). We found that: (a) TnC in solution is flexible; (b) the calcium switch mechanism proposed for isolated TnC occurs in the troponin complex; (c) cardiac TnC central helix is collapsed in troponin ternary complex. Distance measurements between TnC and TnI revealed that there is no significant re-arrangement of the TnC/TnI interface in the TnC C-domain upon calcium binding; however, there is a large movement of the TnI switch peptide induced by binding of Ca2+. In the presence of calcium the switch peptide interacts with the TnC N-domain, while in the absence of Ca2+ the switch peptide moves closer to the TnI inhibitory region. A subfragment of the muscle motor protein, the myosin head, is alone capable of generating force and translation that results in muscle contraction. The myosin head has two distinct domains: a catalytic domain that binds actin and hydrolyzes ATP and a regulatory domain that functions as a lever. Hydrolysis of ATP and binding to actin induces strain within the upper and lower halves of the catalytic domains that is then amplified by the lever arm action of the regulatory domain. For this mechanism to be operative two things should hold: (a) the cleft between upper and lower domains of the catalytic domain closes on binding to actin and (b) the regulatory domain is able to move with respect to the catalytic domain. The first point, closure of the actin binding cleft upon binding of myosin to actin, was investigated using dipolar and pulsed-EPR experiments that are sensitive enough to to measure distances between spin labels placed on either site of the actin binding cleft. As hypothesized, the distances across the cleft were smaller in the presence of actin than in its absence. Movement of the regulatory domain was addressed by determining the domain dynamics of smooth muscle myosin using saturation transfer EPR and phosphorescence anisotropy. Results from these experiments revealed that the regulatory domain is 20-fold more restricted in motion than the catalytic domain implying that a hinge exists between the two domains. It is likely that the inhibition of the regulatory domain dynamics as compared to the skeletal muscle myosin correlates with the inhibited state of the smooth muscle myosin ATPase. Show less

Unraveling the detailed molecular mechanisms involved in muscle contraction is a prerequisite for understanding the molecular basis of muscular disorders. Troponin (Tn) plays a key role in muscle regulation, and it is composed of three subunits: TnC, TnI and TnT. This study is aimed at understanding conformational changes occurring in TnI during muscle activation and force generation. The primary experimental technique used is site specific spin labeling combined with electron paramagnetic... Show moreUnraveling the detailed molecular mechanisms involved in muscle contraction is a prerequisite for understanding the molecular basis of muscular disorders. Troponin (Tn) plays a key role in muscle regulation, and it is composed of three subunits: TnC, TnI and TnT. This study is aimed at understanding conformational changes occurring in TnI during muscle activation and force generation. The primary experimental technique used is site specific spin labeling combined with electron paramagnetic resonance (SDSL-EPR). In present study, we used SDSL EPR technique to observe the orientational changes of TnI-TnT coiled-coil domain and the conformational changes of TnI C-terminal domain between different thin filament states. The fiber tilts spectra of TnI133-MTSSL reconstituted fibers showed that in this domain, TnI is well ordered and oriented at a specific angle with respect to the actin filament. After simulations using a nonlinear-least-squares (NLS) approach, we found that TnI-TnT coiled-coil undergoes a significant orientational change between different states: from "Blocked" to "Closed" state, TnI re-orients itself ~50° axially; from "Closed" to "Open" state, TnI re-orients itself 90° azimuthally. In order to examine the conformational change of TnI C-terminal region upon Ca2+ binding, DEER distance measurements between two residues in TnI C-terminal region (residue 170 and residue 202) were performed. The broad distance distribution between residue 170 and 202 indicates that the TnI C-terminal region is flexible. Unlike TnC and other regions of TnI, in solution, this region is not affected by Ca2+ binding. The reconstitution of the troponin complex to Tm/actin does not change the flexibility of the TnI C-terminal region. By using both DEER and FRET techniques to monitor the distance change between TnI and actin upon Ca2+ binding, we discovered that this region shifts away from actin in thin filaments when Ca2+ is bound. This is the first direct observation of such a conformational change. Show less

Although theory indicates that indirect genetic benefits through mate choice should be widespread, empirical work has often either failed to detect such benefits or shown a net cost to the presence of sexual selection. In this dissertation, I test whether sexual selection facilitates adaptation in populations of Drosophila melanogaster by using an experimental evolution approach where mating system is manipulated. In chapter two, I tested whether sexual selection increases the speed with... Show moreAlthough theory indicates that indirect genetic benefits through mate choice should be widespread, empirical work has often either failed to detect such benefits or shown a net cost to the presence of sexual selection. In this dissertation, I test whether sexual selection facilitates adaptation in populations of Drosophila melanogaster by using an experimental evolution approach where mating system is manipulated. In chapter two, I tested whether sexual selection increases the speed with which a conditionally deleterious allele is removed from a laboratory population of Drosophila melanogaster. Loss of the deleterious allele was more rapid when sexual selection was allowed to act. I also quantified the strength of both nonsexual and sexual selection against the deleterious allele using maximum likelihood estimation. In contrast to recent experiments employing monogamy/polygamy designs, our results demonstrate a population-level benefit to sexual selection that is consistent with the operation of good-genes female choice. If most mutations are deleterious to both overall fitness and condition-dependent traits affecting mating success, sexual selection will purge mutation load and increase nonsexual fitness. In chapter three, I explored this possibility with populations of Drosophila melanogaster exhibiting artificially-elevated levels of deleterious variation and evolving in the presence or absence of sexual selection. After 60 generations of experimental evolution, monogamous populations exhibited higher total reproductive output than polygamous populations. Parental environment also affected fitness measures—flies that evolved in the presence of sexual conflict exhibited reduced nonsexual fitness when their parents experienced a polygamous environment, indicating trans-generational effects of male harassment and highlighting the importance of a common garden design. This cost of parental promiscuity was nearly absent in monogamous lines, evidence for the evolution of reduced sexual antagonism. There was no evidence for an overall difference in egg-to-adult viability between treatments. If mutation load was reduced by the action of sexual selection in this experiment, the resultant gain in fitness was not sufficient to overcome the costs of sexual antagonism. In chapter four, I examined the transcription profiles of these long-term monogamous and polygamous populations. In particular, I looked at two broad classes of sexually-selected genes: those encoding male seminal fluid proteins (Sfps) and female proteases. There is evidence for rapid evolution and sexual conflict in both of these sets of genes. In monogamous populations, Sfps show reduced expression overall, consistent with the idea that they either represent a wasteful use of resources in monogamous populations, due to the absence of male-male competition, and/or harm females. The regulatory response in mated females is in line with these results, as polygamous females upregulate expression of proteases while monogamous females downregulate expression. Expression patterns in virgin females, however, show a conflicting pattern as females from monogamous populations express proteases at a higher basal level. The results of these projects address a major question about the adaptive value of sexual selection. Clearly, sexual selection can accelerate adaptation by purging deleterious alleles. On balance, however, sexual selection depresses nonsexual fitness in polygamous populations when compared to monogamous populations that do not experience sexual conflict, and there is evidence that this decline is due to antagonism between the sexes. Show less

Large-scale cytogenetic mapping in maize has been a major challenge primarily due to fact that the maize genome has a low gene density and an abundance of repetitive sequence elements. Using a unique combination of biological and genome resources for maize, sorghum, and oat, we have solved this problem and produced a cytogenetic FISH map of maize pachytene-stage chromosome 9 with 32 maize markers. The genetically mapped markers used are distributed along the linkage maps at an average spacing... Show moreLarge-scale cytogenetic mapping in maize has been a major challenge primarily due to fact that the maize genome has a low gene density and an abundance of repetitive sequence elements. Using a unique combination of biological and genome resources for maize, sorghum, and oat, we have solved this problem and produced a cytogenetic FISH map of maize pachytene-stage chromosome 9 with 32 maize markers. The genetically mapped markers used are distributed along the linkage maps at an average spacing of 5 centiMorgans. Each locus was mapped by means of multicolor direct FISH with a fluorescently labeled probe mix containing a whole chromosome paint, a single sorghum BAC clone, and the centromeric sequence, CentC. A maize-chromosome-addition line of oat was used for bright unambiguous identification of the maize 9 fiber within pachytene chromosome spreads. The locations of the sorghum BAC FISH signals were determined, and each new cytogenetic locus was assigned a centiMcClintock position on the short (9S) or long (9L) arm. Nearly all of the markers appeared in the same order on linkage and cytogenetic maps but at different relative positions on the two. The CentC FISH signal was localized between tda66 (at 9S.03) and cdo17 (at 9L.03). Several regions of genome hyperexpansion on maize chromosome 9 were found by comparative analysis of relative marker spacing in maize and sorghum. This transgenomic cytogenetic FISH map creates anchors between various maps of maize and sorghum integrating genetic markers, BAC fingerprints, and BAC hybridization data. It will provide tools for validating sequenced genomes, a foundation for exploring genomic diversity among related species, and a framework for comparative mapping of other plants with large and complex genomes. Show less

Competition for shared consumable resources is an important form of competition and has been investigated using mechanistic models of consumer and resource growth. The shape of the relationship between consumption and resource density has traditionally been linear but there are many biological phenomena that can lead to nonlinear relationships between consumption and resource density. This nonlinearity has been shown to have important effects for competition between two consumers for a single... Show moreCompetition for shared consumable resources is an important form of competition and has been investigated using mechanistic models of consumer and resource growth. The shape of the relationship between consumption and resource density has traditionally been linear but there are many biological phenomena that can lead to nonlinear relationships between consumption and resource density. This nonlinearity has been shown to have important effects for competition between two consumers for a single limiting resource, yet our understanding of multi species competition for multiple resources has been based mostly on results from models with a linear function describing consumption. The mechanism of coexistence in these models is resource partitioning, a central concept for both competition and coexistence. I evaluate how nonlinear relationships between consumption and resource density affect expectations of coexistence by directly comparing analytical results from MacArthur style consumer-resource models with linear functional responses and nonlinear functional responses. The general concepts defining resource partitioning are the same for all models that I analyze; the ratio of per-capita resource growth must be between the ratios of proportional resource use at equilibrium for each consumer. Nonlinearity affects the way in which consumers draw down resources to equilibrium compared to the linear model. These effects are additive when consumptive rate saturates with total density and both additive and multiplicative when consumptive rate saturates independently on each resource. Interesting and unintuitive cases arise when consumers are limited at high resource densities including negative R*s that lead to coexistence. When consumptive rate saturates with total resource density, inhibition of consumption of one resource by consumption of others can buffer competitive advantages of species and may produce more regions of coexistence than when consumptive rate saturates on each resource independently. Future studies investigating resource competition will need to measure a multidimensional functional response that manipulates both total and individual resource density in a response surface design to uncover possible conditions for coexistence that before this study had previously not been considered. Show less

Date Issued

2012

Identifier

FSU_migr_etd-4957

Format

Thesis

Title

Control of Prolactin Secretion by Central Oxytocin in Cervically Stimulated Ovariectomized Rats.

Prolactin is a protein hormone predominately synthesized in and secreted from cells in the anterior pituitary gland called lactotrophs. Although prolactin is elevated during other physiological states, mating induces a unique pattern of prolactin secretion. Prolactin is elevated twice a day for several days in response to a mating stimulus, suggesting a mnemonic. The neural pathway in which the mating stimulus initiates this unique prolactin secretion is unknown. However, hypothalamic... Show moreProlactin is a protein hormone predominately synthesized in and secreted from cells in the anterior pituitary gland called lactotrophs. Although prolactin is elevated during other physiological states, mating induces a unique pattern of prolactin secretion. Prolactin is elevated twice a day for several days in response to a mating stimulus, suggesting a mnemonic. The neural pathway in which the mating stimulus initiates this unique prolactin secretion is unknown. However, hypothalamic inhibitory neuroendocrine dopamine neurons and stimulatory oxytocin neurons are demonstrated to be involved. Our laboratory has shown that a peripheral injection of oxytocin initiates prolactin surges like those induced by mating. This suggests that oxytocin may initiate the mating-induced prolactin surges. Whether the bolus injection of oxytocin initiated these surges by acting peripherally or centrally was investigated in this dissertation. Results showed that oxytocin does not initiate these surges by acting peripherally since a peripheral oxytocin antagonist did not block the initiation of the surges. However, peripheral oxytocin is important for maintaining the surges. Therefore, central oxytocin must initiate twice daily prolactin surges, likely from the hypothalamic paraventricular nucleus. Since the mating stimulus must convey its signal from the uterine cervix to the hypothalamus a pathway was investigated. A relay of the mating stimulus via brainstem noradrenergic input to the paraventricular nucleus was shown. Only brainstem noradrenergic neurons that projected to paraventricular nucleus were activated by the mating stimulus, suggesting a specific pathway for the initiation of mating-induced prolactin surges. In addition, norepinephrine was released in the paraventricular nucleus in response to a mating stimulus. Lastly, oxytocin neurons in the hypothalamic paraventricular nucleus were directly investigated. Surprisingly, hypothalamic oxytocin neurons were not acutely activated by a mating stimulus, suggesting that the paraventricular nucleus is not a source of oxytocin important in initiating the mating-induced prolactin surges. Show less

Electron microscopy is an important technique for observing macromolecular structures, such as DNA and viruses, which would be too small to see under light microscopy. This type of microscopy utilizes electrons as its illumination source, produced by an electron gun, to generate an image of the specimen which is captured by either a charged coupled device (CCD) or direct electron detector (DED) camera. Specimens in electron microscopy can either be stained with heavy atom, embedded in plastic... Show moreElectron microscopy is an important technique for observing macromolecular structures, such as DNA and viruses, which would be too small to see under light microscopy. This type of microscopy utilizes electrons as its illumination source, produced by an electron gun, to generate an image of the specimen which is captured by either a charged coupled device (CCD) or direct electron detector (DED) camera. Specimens in electron microscopy can either be stained with heavy atom, embedded in plastic, or embedded in vitrified ice. Cryo-electron microscopy (cryo-EM) embeds specimens in a vitreous ice layer that resembles the specimen’s natural environment and increases the overall resolution of the specimen. In conjunction with cryo-electron tomography (cryo-ET), cryo-EM specimens can be tilted on a specimen holder to collect multiple 2D views in order to generate a 3D reconstruction through a weighted back-projection algorithm. The projections are first corrected to counter the effects of contrast transfer function (CTF), which can filter out high resolution information. The resulting tomogram undergoes cycles of image processing steps such as multivariate data analysis, classification, and subvolume averaging to bring out the features of the specimen. At the borders of the striated muscle sarcomere, there exists an electron dense structure called the Z-Disc. The arrangement of thin filaments in the Z-Disc differs between vertebrates and invertebrates. Z-Discs of vertebrates have a tetragonal lattice that contrasts with the hexagonal lattice seen in the A-Band, which might be caused by arrangement of an elastic protein named Titin from the A-Band to the Z-Disc. The tetragonal lattice in the vertebrate Z-Disc has two structural states, small-square and basket-weave, depending on the contraction state of the muscle. Invertebrate Z-Discs have a hexagonal lattice that contains five connecting densities that form large and small solvent channels. Z-Discs contain many proteins that are important for structural stability and signaling functions. Three Z-Disc proteins that are structurally important in invertebrate Z-Discs are α-actinin, an actin crosslinker, Kettin and Projectin, the latter being components of the elastic connecting filament. Alternative Z-Disc isolation methods were explored using Wild-type (WT) Drosophila and Sls-RNAi knockdown Drosophila for the possibility of using the specimens for cryo-EM. The insect flight muscle (IFM) was dissected from the thorax of WT Drosophila and the individual myofibrils were obtained through a homogenization and cleaning process. The Z-Discs were isolated from the myofibrils by exposing them to high salt buffers, to remove thick filaments, and gelsolin, to remove thin filaments. The isolated Z-Discs were negatively stained and observed under an electron microscope. The lattice arrangement of the thin filaments could not be seen due to the stain, but this method produced many Z-Discs on the EM grids. Cryo-EM samples of the isolated WT Drosophila Z-Discs could not be obtained due to problems pertaining to the plunge freezing method. Sls-RNAi Drosophila was obtained using the GAL4/UAS method to generate smaller Z-Discs and decrease the width of the myofibrils due to a decrease in the presence of Kettin. The IFM was extracted from the thorax, but the myofibrils were not exposed to high salt buffers and gelsolin. Under negative stain, the myofibrils observed produced Z-Discs about 500 nm in width, which is ideal for cryo-ET conditions. However, a width of 200-300 nm would produce higher resolution images for a 3D reconstruction. A cryo-EM 3D reconstruction was generated from isolated Lethocerus Z-Discs to confirm the structural features seen in plastic-embedded sections of Apis. Projections for cryo-ET were collected using a DED camera and underwent CTF correction. The tilt series images were coarse-aligned and went through cycles of refinement using an Appion-based database with Protomo. A 3D reconstruction was generated using a weighted back-projection algorithm, filtered to bring out structural features (subvolumes), and then the subvolumes were averaged through single- and multi-reference alignment. The results were visualized in CHIMERA which confirmed the hexagonal lattice arrangement of thin filaments as reported previously in Apis Z-Discs. The location of connecting densities, C1 and C2, were confirmed as forming apices and bases of the large solvent channel, while C3 and C5 were confirmed to be connecting thin filaments of opposite orientation at the tapered end of the small solvent channel. C4 connecting density/three-wheel spoke was seen linking the ends of three thin filaments in the same orientation that form the small solvent channel. C1 and C2 were proposed to contain α-actinin, especially in C2 where an atomic model of F-actin with CH1 domains closely interacted with an atomic model of α-actinin in the C2 density map. The results of this experiment confirmed what was currently known about the invertebrate Z-Disc structure, but the locations of Z-Disc proteins, Kettin and Projectin, are yet to be determined. Show less

The work presented in this dissertation represents investigations into the mechanism of two neurological disorders. The first set of experiments was aimed at examining the morphology of post-synaptic structures called dendritic spines in a mouse model of Down's syndrome. The second set of experiments was aimed at examining the regional pattern and cellular distribution of possible therapeutic targets, histone deacetylases, in treating the symptoms of Huntington's disease. In the first set of... Show moreThe work presented in this dissertation represents investigations into the mechanism of two neurological disorders. The first set of experiments was aimed at examining the morphology of post-synaptic structures called dendritic spines in a mouse model of Down's syndrome. The second set of experiments was aimed at examining the regional pattern and cellular distribution of possible therapeutic targets, histone deacetylases, in treating the symptoms of Huntington's disease. In the first set of experiments, we examined a mouse model of Down's syndrome, the Ts65Dn mouse, to determine if it mimics the dendritic spine abnormalities in area CA1 of hippocampus that have been documented in human individuals with the disorder. The Ts65Dn mouse represents a partial trisomy of the murine chromosome homologous to a large portion of human chromosome 21, which is present in 3 copies rather than 2 in Down's syndrome. These mice show behavioral abnormalities and learning deficits that are thought to replicate the mental retardation that is a prominent characteristic of Down's syndrome. As many of the behavioral and learning paradigms used to test these mice require hippocampal function, and neurons from hippocampal tissue taken from Down's syndrome individuals show a reduction in the density of dendritic spines, we sought to determine if Ts65Dn mice exhibit the same morphological abnormality. Dendritic spine densities on the apical branches of CA1 hippocampal neurons in Ts65Dn animals were not significantly different when compared to those from euploid (normal chromosome number) littermates. In addition, morphological analysis of dendritic spine shape demonstrated that the proportion of dendritic spines in each of the four major spine shape categories (stubby, thin, filopodia and mushroom) was not different between the two conditions. As the environment for cells in a cultured slice are most likely very different from those experienced in the intact animal, we examined if neurons in the intact brain exhibited signs of abnormal dendritic spine density in the Ts65Dn mouse. Analysis of dendritic spine densities on apical branches of CA1 hippocampal neurons from mice sacrificed at 2 weeks, 3 months or 6 months of age showed no significant differences between the euploid and trisomic conditions. Furthermore, Western blot analysis showed that Ts65Dn do not have reduced expression of a dendritic spine protein, drebrin, as has been reported in Down's syndrome. The second set of experiments that are described concern the immunohistochemical localization of two enzymes involved in the transcriptional regulation of genes. These enzymes, called histone deacetylases, or HDACs, aid in the regulation of histone acetylation levels as a mechanism to control access of transcription factors to gene sequences. Recently, it was found that compounds that inhibit enzymes that remove acetyl groups demonstrate therapeutic effects in animal models of Huntington's disease, a neurodegenerative disorder that targets brain regions important for movement control. The work presented here describes the immunohistochemical localization of a HDAC2 and HDAC7. HDAC7 immunohistochemistry was consistent with biochemical studies demonstrating that HDAC7 can be present in both the cytoplasm and the nucleus. A finding of interest is that not all neurons of the murine brain express HDAC7, nor is it localized to the same subcellular compartment in all cell types. Granule cells of the hippocampal dentate gyrus and of the cerebellum showed very little immunoreactivity for HDAC7. Apical dendrites of the CA1 and CA3 regions of hippocampus showed very heavy cytoplasmic staining. Deeper cortical layers showed pyramidal neurons with heavier staining than superficial pyramidal layers in almost all cortical regions, except for the orbital, insular and piriform cortices which showed heavy staining in superficial layers as well. In general, staining of the olfactory system appeared more intense than other sensory system regions. Fibers along the striatonigral bundle, from the caudate to the substantia nigra reticulata showed heavy immunoreactivity. These data suggest that targeting therapeutics to HDAC7 activity may indeed be useful, as the striatonigral pathway is a key component of movement control. HDAC2 immunoreactivity confirmed that HDAC2 is strictly a nuclear localized protein. In addition, it appeared that HDAC2 is ubiquitously expressed throughout the murine brain in all brain regions. Interestingly, HDAC2 staining occurred only in neurons that showed NeuN staining, demonstrating that it is a neuron-specific protein. Therefore, therapeutic inhibition of HDACs that include inhibition of HDAC2 may affect many aspects of neurologic functioning in multiple brain regions. It is our hope that this work will provide some added detail and knowledge to the greater neuroscience community, and will aid in the greater understanding of the organization and function of the brain and ultimately aid in development of treatments for and ultimately the cure of neurological diseases and disorders. Show less

Dental morphology reflects an organism's diet. Functional dental theory predicts that tooth shape corresponds to the mechanical properties of food. Most studies of mammalian teeth have focused on qualitative measures of dental anatomy and have not formally tested how the functional components of teeth adapt in response to diet. Here, murine rodents (Old World rats and mice) are used to quantify the relationship between diet and tooth shape and further, to identify a suite of functional dental... Show moreDental morphology reflects an organism's diet. Functional dental theory predicts that tooth shape corresponds to the mechanical properties of food. Most studies of mammalian teeth have focused on qualitative measures of dental anatomy and have not formally tested how the functional components of teeth adapt in response to diet. Here, murine rodents (Old World rats and mice) are used to quantify the relationship between diet and tooth shape and further, to identify a suite of functional dental characters that best distinguish amongst diets. Phylogenetic relationships are also taken into consideration. Biomechanically based size and shape predictions were also developed. Eighty-eight dental characters were extracted from images of the incisors and upper and lower tooth rows for 98 species. Results show that species with plant-dominated diets typically have deep incisors, a longer M3, longer molar crests, blunt posteriorly angled cusps, and more expanded laterally oriented occlusal cusps than species with animal dominated diets. Measures of incisor depth, crest length, cusp angle and sharpness, occlusal cusp orientation, M3/m3 length, most effectively separate amongst diets. Results suggest that these specific aspects of dental morphology can be used to accurately infer diet for both extinct and extant murine species. After performing a phylogentically independent analysis, my results suggest that phylogenetic history does not constrain dental morphology and convergent dental adaptations arise in response to diet. Show less